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We all know deoxyribonucleotides form DNA while ribonucleotides form RNA, DNA is double stranded while RNA is single stranded, and RNA can transcribe from DNA. We also know that DNA use A, G, C, T, while RNA use A, G, C, U and that it's the DNA that stores genetics information, not RNA.
My question is: how does the lost oxygen atom on deoxyribonucleotides 2' site of the sugar (turning it into a ribonucleotide), make such a large difference in function? It just an oxygen atom. Why don't they just all use U or all use T?
Why deoxyribose for DNA and ribose for RNA? This answer may solve the question why is DNA double strand, RNA single strand, but it doesnt explain why does RNA uses U instead of T.
For the switch from thymine (T) to uracil (U), it actually doesn't have anything to do with the sugar backbone of the nucleotides. This article on Science Daily explains that U is more cost efficient to produce than T because it can be easily gotten from a chemical degradation of cytosine (C). Since RNA has a short life span and because the cell needs a lot of RNA it uses U. DNA, on the other hand, undergoes a lot of checks and repairs. If it were to use U, it wouldn't know if the U was really a U or if it was a C that got damaged. Since it is important to keep the number of mutations to a minimum, DNA uses the more costly T to ensure fidelity.
I would also like to point out @AMR's comment. It isn't just an oxygen atom. An oxygen atom is the difference between the air we breath and ozone that can kill you. It's the difference between water and highly flammable hydrogen gas.
Compare the Phosphates Sugars and Bases of DNA and RNA
DNA and RNA are nucleic acids, which are basically made up of a nitrogenous base containing pentose sugars linked via phosphate groups. The building blocks of nucleic acids are called nucleotides. Nucleic acids serve as the cell’s genetic material by storing information, which is required for the development, functioning, and reproduction of organisms. Most organisms use DNA as their genetic material, while few of them like retroviruses use RNA as their genetic material. DNA is stable when compared to RNA due to the differences in phosphate sugars and bases shared by each of them. One, two or three phosphate groups can be attached to the pentose sugar, producing mono-, di- and triphosphates respectively. Pentose sugar used by DNA is deoxyribose and the pentose sugar used by RNA is ribose. Nitrogenous bases found in DNA are adenine, guanine, cytosine and thymine. In RNA, thymine is replaced by uracil.
1. What are Phosphates
2. What are Sugars
3. What are Bases
4. Comparison of Phosphates Sugars and Bases of DNA and RNA
A DNA strand is a polymer of nucleoside monophosphates held together by phosphodiester bonds. Two such paired strands make up the DNA molecule, which is then twisted into a helix. In the most common Bform, the DNA helix has a repeat of 10.5 base pairs per turn, with sugars and phosphate forming the covalent phosphodiester &ldquobackbone&rdquo of the molecule and the adenine, guanine, cytosine, and thymine bases oriented in the middle where they form the now familiar base pairs that look like the rungs of a ladder.
The term nucleotide refers to the building blocks of both DNA (deoxyribonucleoside triphosphates, dNTPs) and RNA (ribonucleoside triphosphates, NTPs). In order to discuss this important group of molecules, it is necessary to define some terms.
Nucleotides contain three primary structural components. These are a nitrogenous base, a pentose sugar, and at least one phosphate. Molecules that contain only a sugar and a nitrogenous base (no phosphate) are called nucleosides. The nitrogenous bases found in nucleic acids include adenine and guanine (called purines) and cytosine, uracil, or thymine (called pyrimidines). There are two sugars found in nucleotides - deoxyribose and ribose (Figure 2.128). By convention, the carbons on these sugars are labeled 1&rsquo to 5&rsquo. (This is to distinguish the carbons on the sugars from those on the bases, which have their carbons simply labeled as 1, 2, 3, etc.) Deoxyribose differs from ribose at the 2&rsquo position, with ribose having an OH group, where deoxyribose has H.
Figure 2.128 - Nucleotides, nucleosides, and bases
Nucleotides containing deoxyribose are called deoxyribonucleotides and are the forms found in DNA. Nucleotides containing ribose are called ribonucleotides and are found in RNA. Both DNA and RNA contain nucleotides with adenine, guanine, and cytosine, but with very minor exceptions, RNA contains uracil nucleotides, whereas DNA contains thymine nucleotides. When a base is attached to a sugar, the product, a nucleoside, gains a new name.
- uracil-containing = uridine (attached to ribose) / deoxyuridine (attached to deoxyribose)
- thymine-containing = ribothymidine (attached to ribose) / thymidine (attached to deoxyribose)
- cytosine-containing = cytidine (attached to ribose - Figure 2.129) / deoxycytidine (attached to deoxyribose)
- guanine-containing = guanosine (attached to ribose) / deoxyguanosine (attached to deoxyribose)
- adenine-containing = adenosine (attached to ribose) / deoxyadenosine (attached to deoxyribose)
Of these, deoxyuridine and ribothymidine are the least common. The addition of one or more phosphates to a nucleoside makes it a nucleotide. Nucleotides are often referred to as nucleoside phosphates, for this reason. The number of phosphates in the nucleotide is indicated by the appropriate prefixes (mono, di or tri).
Figure 2.129 Cytidine
Thus, cytidine, for example, refers to a nucleoside (no phosphate), but cytidine monophosphate refers to a nucleotide (with one phosphate). Addition of second and third phosphates to a nucleoside monophosphate requires energy, due to the repulsion of negatively charged phosphates and this chemical energy is the basis of the high energy triphosphate nucleotides (such as ATP) that fuel cells.
Note: Ribonucleotides as Energy Sources
Though ATP is the most common and best known cellular energy source, each of the four ribonucleotides plays important roles in providing energy. GTP, for example, is the energy source for protein synthesis (translation) as well as for a handful of metabolic reactions. A bond between UDP and glucose makes UDP-glucose, the building block for making glycogen. CDP is similarly linked to several different molecular building blocks important for glycerophospholipid synthesis (such as CDP-diacylglycerol).
The bulk of ATP made in cells is not from directly coupled biochemical metabolism, but rather by the combined processes of electron transport and oxidative phosphorylation in mitochondria and/or photophosphorylation that occurs in the chloroplasts of photosynthetic organisms. Triphosphate energy in ATP is transferred to the other nucleosides/nucleotides by action of enzymes called kinases. For example, nucleoside diphosphokinase (NDPK) catalyzes the following reaction
where &lsquoN&rsquo of &ldquoNDP&rdquo and &ldquoNTP corresponds to any base. Other kinases can put single phosphates onto nucleosides or onto nucleoside monophosphates using energy from ATP.
Individual deoxyribonucleotides are derived from corresponding ribonucleoside diphosphates via catalysis by the enzyme known as ribonucleotide reductase (RNR). The deoxyribonucleoside diphosphates are then converted to the corresponding triphosphates (dNTPs) by the addition of a phosphate group. Synthesis of nucleotides containing thymine is distinct from synthesis of all of the other nucleotides and will be discussed later.
Building DNA strands
Each DNA strand is built from dNTPs by the formation of a phosphodiester bond, catalyzed by DNA polymerase, between the 3&rsquoOH of one nucleotide and the 5&rsquo phosphate of the next. The result of this directional growth of the strand is that the one end of the strand has a free 5&rsquo phosphate and the other a free 3&rsquo hydroxyl group (Figure 2.130). These are designated as the 5&rsquo and 3&rsquo ends of the strand.
Figure 2.130 - 5&rsquo-3&rsquo Polarity of a DNA strand
Figure 2.131 shows two strands of DNA (left and right). The strand on the left, from 5&rsquo to 3&rsquo reads T-C-G-A, whereas the strand on the right, reading from 5&rsquo to 3&rsquo is T-C-G-A. The strands in a double-stranded DNA are arranged in an anti-parallel fashion with the 5&rsquo end of one strand across from the 3&rsquo end of the other.
Purine and Pyrimidine Metabolism
One of the important specialized pathways of a number of amino acids is the synthesis of purine and pyrimidine nucleotides. These nucleotides are important for a number of reasons. Most of them, not just ATP, are the sources of energy that drive most of our reactions. ATP is the most commonly used source but GTP is used in protein synthesis as well as a few other reactions. UTP is the source of energy for activating glucose and galactose. CTP is an energy source in lipid metabolism. AMP is part of the structure of some of the coenzymes like NAD and Coenzyme A. And, of course, the nucleotides are part of nucleic acids. Neither the bases nor the nucleotides are required dietary components. (Another perspective on this.) We can both synthesize them de novo and salvage and reuse those we already have.
There are two kinds of nitrogen-containing bases - purines and pyrimidines. Purines consist of a six-membered and a five-membered nitrogen-containing ring, fused together. Pyridmidines have only a six-membered nitrogen-containing ring. There are 4 purines and 4 pyrimidines that are of concern to us.
- Adenine = 6-amino purine
- Guanine = 2-amino-6-oxy purine
- Hypoxanthine = 6-oxy purine
- Xanthine = 2,6-dioxy purine
Adenine and guanine are found in both DNA and RNA. Hypoxanthine and xanthine are not incorporated into the nucleic acids as they are being synthesized but are important intermediates in the synthesis and degradation of the purine nucleotides.
- Uracil = 2,4-dioxy pyrimidine
- Thymine = 2,4-dioxy-5-methyl pyrimidine
- Cytosine = 2-oxy-4-amino pyrimidine
- Orotic acid = 2,4-dioxy-6-carboxy pyrimidine
Cytosine is found in both DNA and RNA. Uracil is found only in RNA. Thymine is normally found in DNA. Sometimes tRNA will contain some thymine as well as uracil.
If a sugar, either ribose or 2-deoxyribose , is added to a nitrogen base, the resulting compound is called a nucleoside . Carbon 1 of the sugar is attached to nitrogen 9 of a purine base or to nitrogen 1 of a pyrimidine base. The names of purine nucleosides end in -osine and the names of pyrimidine nucleosides end in -idine. The convention is to number the ring atoms of the base normally and to use l', etc. to distinguish the ring atoms of the sugar. Unless otherwise specificed, the sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d- is placed before the name.
- Inosine - the base in inosine is hypoxanthine
Adding one or more phosphates to the sugar portion of a nucleoside results in a nucleotide . Generally, the phosphate is in ester linkage to carbon 5' of the sugar. If more than one phosphate is present, they are generally in acid anhydride linkages to each other. If such is the case, no position designation in the name is required. If the phosphate is in any other position, however, the position must be designated. For example, 3'-5' cAMP indicates that a phosphate is in ester linkage to both the 3' and 5' hydroxyl groups of an adenosine molecule and forms a cyclic structure. 2'-GMP would indicate that a phosphate is in ester linkage to the 2' hydroxyl group of a guanosine. Some representative names are:
- AMP = adenosine monophosphate = adenylic acid
- CDP = cytidine diphosphate
- dGTP = deoxy guanosine triphosphate
- dTTP = deoxy thymidine triphosphate (more commonly designated TTP)
- cAMP = 3'-5' cyclic adenosine monophosphate
Nucleotides are joined together by 3'-5' phosphodiester bonds to form polynucleotides. Polymerization of ribonucleotides will produce an RNA while polymerization of deoxyribonucleotides leads to DNA.
Hydrolysis of Polynucleotides
Most, but not all, nucleic acids in the cell are associated with protein. Dietary nucleoprotein is degraded by pancreatic enzymes and tissue nucleoprotein by lysosomal enzymes. After dissociation of the protein and nucleic acid, the protein is metabolized like any other protein.
The nucleic acids are hydrolyzed randomly by nucleases to yield a mixture of polynucleotides. These are further cleaved by phosphodiesterases (exonucleases) to a mixture of the mononucleotides. The specificity of the pancreatic nucleotidases gives the 3'-nucleotides and that of the lysosomal nucleotidases gives the biologically important 5'-nucleotides.
The nucleotides are hydrolyzed by nucleotidases to give the nucleosides and P i . This is probably the end product in the intestine with the nucleosides being the primary form absorbed. In at least some tissues, the nucleosides undergo phosphorolysis with nucleoside phosphorylases to yield the base and ribose 1-P (or deoxyribose 1-P). Since R 1-P and R 5-P are in equilibrium, the sugar phosphate can either be reincorporated into nucleotides or metabolized via the Hexose Monophosphate Pathway. The purine and pyrimidine bases released are either degraded or salvaged for reincorporation into nucleotides. There is significant turnover of all kinds of RNA as well as the nucleotide pool. DNA doesn't turnover but portions of the molecule are excised as part of a repair process.
Purine and pyrimidines from tissue turnover which are not salvaged are catabolized and excreted. Little dietary purine is used and that which is absorbed is largely catabolized as well. Catabolism of purines and pyrimidines occurs in a less useful fashion than did the catabolism of amino acids in that we do not derive any significant amount of energy from the catabolism of purines and pyrimidines. Pyrimidine catabolism, however, does produce beta-alanine, and the endproduct of purine catabolism, which is uric acid in man, may serve as a scavenger of reactive oxygen species.
Nucleotides to Bases
Guanine nucleotides are hydrolyzed to the nucleoside guanosine which undergoes phosphorolysis to guanine and ribose 1-P . Man's intracellular nucleotidases are not very active toward AMP, however. Rather, AMP is deaminated by the enzyme adenylate (AMP) deaminase to IMP . In the catobilsm of purine nucleotides, IMP is further degraded by hydrolysis with nucleotidase to inosine and then phosphorolysis to hypoxanthine .
Adenosine does occur but usually arises from S-Adenosylmethionine during the course of transmethylation reactions. Adenosine is deaminated to inosine by an adenosine deaminase. Deficiencies in either adenosine deaminase or in the purine nucleoside phosphorylase lead to two different immunodeficiency diseases by mechanisms that are not clearly understood. With adenosine deaminase deficiency , both T and B-cell immunity is affected. The phosphorylase deficiency affects the T cells but B cells are normal. In September, 1990, a 4 year old girl was treated for adenosine deaminase deficiency by genetically engineering her cells to incorporate the gene. The treatment,so far, seems to be successful.
Whether or not methylated purines are catabolized depends upon the location of the methyl group. If the methyl is on an -NH 2 , it is removed along with the -NH 2 and the core is metabolized in the usual fashion. If the methyl is on a ring nitrogen, the compound is excreted unchanged in the urine.
Bases to Uric Acid
Both adenine and guanine nucleotides converge at the common intermediate xanthine . Hypoxanthine, representing the original adenine, is oxidized to xanthine by the enzyme xanthine oxidase . Guanine is deaminated, with the amino group released as ammonia, to xanthine. If this process is occurring in tissues other than liver, most of the ammonia will be transported to the liver as glutamine for ultimate excretion as urea.
Xanthine, like hypoxanthine, is oxidized by oxygen and xanthine oxidase with the production of hydrogen peroxide. In man, the urate is excreted and the hydrogen peroxide is degraded by catalase. Xanthine oxidase is present in significant concentration only in liver and intestine. The pathway to the nucleosides, possibly to the free bases, is present in many tissues.
Gouts and Hyperuricemia
Both undissociated uric acid and the monosodium salt (primary form in blood) are only sparingly soluble. The limited solubility is not ordinarily a problem in urine unless the urine is very acid or has high [Ca 2+ ]. [Urate salts coprecipitate with calcium salts and can form stones in kidney or bladder.] A very high concentration of urate in the blood leads to a fairly common group of diseases referred to as gout. The incidence of gout in this country is about 3/1000.
Gout is a group of pathological conditions associated with markedly elevated levels of urate in the blood (3-7 mg/dl normal). Hyperuricemia is not always symptomatic, but, in certain individuals, something triggers the deposition of sodium urate crystals in joints and tissues. In addition to the extreme pain accompanying acute attacks, repeated attacks lead to destruction of tissues and severe arthritic-like malformations. The term gout should be restricted to hyperuricemia with the presence of these tophaceous deposits.
Urate in the blood could accumulate either through an overproduction and/or an underexcretion of uric acid. In gouts caused by an overproduction of uric acid, the defects are in the control mechanisms governing the production of - not uric acid itself - but of the nucleotide precursors. The only major control of urate production that we know so far is the availability of substrates (nucleotides, nucleosides or free bases) .
One approach to the treatment of gout is the drug allopurinol , an isomer of hypoxanthine.
Allopurinol is a substrate for xanthine oxidase, but the product binds so tightly that the enzyme is now unable to oxidized its normal substrate. Uric acid production is diminished and xanthine and hypoxanthine levels in the blood rise. These are more soluble than urate and are less likely to deposit as crystals in the joints. Another approach is to stimulate the secretion of urate in the urine.
In summary, all, except ring-methylated, purines are deaminated (with the amino group contributing to the general ammonia pool) and the rings oxidized to uric acid for excretion. Since the purine ring is excreted intact, no energy benefit accrues to man from these carbons.
In order for the rings to be cleaved, they must first be reduced by NADPH . Atoms 2 and 3 of both rings are released as ammonia and carbon dioxide. The rest of the ring is left as a beta-amino acid . Beta-amino isobutyrate from thymine or 5-methyl cytosine is largely excreted. Beta-alanine from cytosine or uracil may either be excreted or incorporated into the brain and muscle dipeptides, carnosine (his-beta-ala) or anserine (methyl his-beta-ala).
Purine and pyrimidine bases which are not degraded are recycled - i.e. reincorporated into nucleotides. This recycling, however, is not sufficient to meet total body requirements and so some de novo synthesis is essential. There are definite tissue differences in the ability to carry out de novo synthesis. De novo synthesis of purines is most active in liver. Non-hepatic tissues generally have limited or even no de novo synthesis. Pyrimidine synthesis occurs in a variety of tissues. For purines, especially, non-hepatic tissues rely heavily on preformed bases - those salvaged from their own intracellular turnover supplemented by bases synthesized in the liver and delivered to tissues via the blood.
"Salvage" of purines is reasonable in most cells because xanthine oxidase, the key enzyme in taking the purines all of the way to uric acid, is significantly active only in liver and intestine. The bases generated by turnover in non-hepatic tissues are not readily degraded to uric acid in those tissues and, therefore, are available for salvage. The liver probably does less salvage but is very active in de novo synthesis - not so much for itself but to help supply the peripheral tissues.
De novo synthesis of both purine and pyrimidine nucleotides occurs from readily available components.
We use for purine nucleotides the entire glycine molecule (atoms 4, 5,7), the amino nitrogen of aspartate (atom 1), amide nitrogen of glutamine (atoms 3, 9), components of the folate-one-carbon pool(atoms 2, 8), carbon dioxide, ribose 5-P from glucose and a great deal of energy in the form of ATP. In de novo synthesis, IMP is the first nucleotide formed. It is then converted to either AMP or GMP.
Since the purines are synthesized as the ribonucleotides, (not as the free bases) a necessary prerequisite is the synthesis of the activated form of ribose 5-phosphate. Ribose 5-phosphate reacts with ATP to form 5-Phosphoribosyl-1-pyrophosphate (PRPP) .
This reaction occurs in many tissues because PRPP has a number of roles - purine and pyrimidine nucleotide synthesis, salvage pathways, NAD and NADP formation. The enzyme is heavily controlled by a variety of compounds (di- and tri-phosphates, 2,3-DPG), presumably to try to match the synthesis of PRPP to a need for the products in which it ultimately appears.
De novo purine nucleotide synthesis occurs actively in the cytosol of the liver where all of the necessary enzymes are present as a macro-molecular aggregate. The first step is a replacement of the pyrophosphate of PRPP by the amide group of glutamine. The product of this reaction is 5-Phosphoribosylamine . The amine group that has been placed on carbon 1 of the sugar becomes nitrogen 9 of the ultimate purine ring. This is the commitment and rate-limiting step of the pathway.
The enzyme is under tight allosteric control by feedback inhibition. Either AMP, GMP, or IMP alone will inhibit the amidotransferase while AMP + GMP or AMP + IMP together act synergistically . This is a fine control and probably the major factor in minute by minute regulation of the enzyme. The nucleotides inhibit the enzyme by causing the small active molecules to aggregate to larger inactive molecules.
[PRPP] also can play a role in regulating the rate. Normal intracellular concentrations of PRPP (which can and do fluctuate) are below the KM of the enzyme for PRPP so there is great potential for increasing the rate of the reaction by increasing the substrate concentration. The kinetics are sigmoidal. The enzyme is not particularly sensitive to changes in [Gln] (Kinetics are hyperbolic and [gln] approximates KM). Very high [PRPP] also overcomes the normal nucleotide feedback inhibition by causing the large, inactive aggregates to dissociate back to the small active molecules.
Purine de novo synthesis is a complex, energy-expensive pathway. It should be, and is, carefully controlled.
Formation of IMP
Once the commitment step has produced the 5-phosphoribosyl amine, the rest of the molecule is formed by a series of additions to make first the 5- and then the 6-membered ring. (Note: the numbers given to the atoms are those of the completed purine ring and names, etc. of the intermediate compounds are not given.) The whole glycine molecule, at the expense of ATP adds to the amino group to provide what will eventually be atoms 4, 5, and 7 of the purine ring (The amino group of 5-phosphoribosyl amine becomes nitrogen N of the purine ring.) One more atom is needed to complete the five-membered ring portion and that is supplied as 5, 10-Methenyl tetrahydrofolate.
Before ring closure occurs, however, the amide of glutamine adds to carbon 4 to start the six-membered ring portion (becomes nitrogen 3). This addition requires ATP. Another ATP is required to join carbon 8 and nitrogen 9 to form the five-membered ring.
The next step is the addition of carbon dioxide (as a carboxyl group) to form carbon 6 of the ring. The amine group of aspartate adds to the carboxyl group with a subsequent removal of fumarate. The amino group is now nitrogen 1 of the final ring. This process, which is typical for the use of the amino group of aspartate, requires ATP. The final atom of the purine ring, carbon 2, is supplied by 10-Formyl tetrahydrofolate. Ring closure produces the purine nucleotide, IMP.
Note that at least 4 ATPs are required in this part of the process. At no time do we have either a free base or a nucleotide.
Formation of AMP and GMP
IMP can then become either AMP or GMP. GMP formation requires that IMP be first oxidized to XMP using NAD. The oxygen at position 2 is substituted by the amide N of glutamine at the expense of ATP. Similarly, GTP provides the energy to convert IMP to AMP . The amino group is provided by aspartate in a mechanism similar to that used in forming nitrogen 1 of the ring. Removal of the carbons of aspartate as fumarate leaves the nitrigen behind as the 6-amino group of the adenine ring. The monophosphates are readily converted to the di- and tri-phosphates.
Control of De Novo Synthesis
Control of purine nucleotide synthesis has two phases. Control of the synthesis as a whole occurs at the amidotransferase step by nucleotide inhibition and/or [PRPP]. The second phase of control is involved with maintaining an appropriate balance (not equality) between ATP and GTP . Each one stimulates the synthesis of the other by providing the energy. Feedback inhibition also controls the branched portion as GMP inhibits the conversion of IMP to XMP and AMP inhibits the conversion of IMP to adenylosuccinate.
One could imagine the controls operating in such a way that if only one of the two nucleotides were required, there would be a partial inhibition of de novo synthesis because of high levels of the other and the IMP synthesized would be directed toward the synthesis of the required nucleotide. If both nucleotides were present in adequate amounts, their synergistic effect on the amidotransferase would result in almost complete inhibition of de novo synthesis.
De Novo Synthesis of Pyrimidine Nucleotides
Since pyrimidine molecules are simpler than purines, so is their synthesis simpler but is still from readily available components. Glutamine's amide nitrogen and carbon dioxide provide atoms 2 and 3 or the pyrimidine ring. They do so, however, after first being converted to carbamoyl phosphate. The other four atoms of the ring are supplied by aspartate. As is true with purine nucleotides, the sugar phosphate portion of the molecule is supplied by PRPP.
Pyrimidine synthesis begins with carbamoyl phosphate synthesized in the cytosol of those tissues capable of making pyrimidines (highest in spleen, thymus, GItract and testes). This uses a different enzyme than the one involved in urea synthesis. Carbamoyl phosphate synthetase II (CPS II) prefers glutamine to free ammonia and has no requirement for N-Acetylglutamate.
Formation of Orotic Acid
Carbamoyl phosphate condenses with aspartate in the presence of aspartate transcarbamylase to yield N-carbamylaspartate which is then converted to dihydroorotate.
In man, CPSII, asp-transcarbamylase, and dihydroorotase activities are part of a multifunctional protein .
Oxidation of the ring by a complex, poorly understood enzyme produces the free pyrimidine, orotic acid. This enzyme is located on the outer face of the inner mitochondrial membrane, in contrast to the other enzymes which are cytosolic. Note the contrast with purine synthesis in which a nucleotide is formed first while pyrimidines are first synthesized as the free base .
Formation of the Nucleotides
Orotic acid is converted to its nucleotide with PRPP. OMP is then converted sequentially - not in a branched pathway - to the other pyrimidine nucleotides. Decarboxylation of OMP gives UMP . O-PRT and OMP decarboxylase are also a multifunctional protein . After conversion of UMP to the triphosphate, the amide of glutamine is added, at the expense of ATP, to yield CTP .
The control of pyrimidine nucleotide synthesis in man is exerted primarily at the level of cytoplasmic CPS II . UTP inhibits the enzyme, competitively with ATP. PRPP activates it. Other secondary sites of control also exist (e.g. OMP decarboxylase is inhibited by UMP and CMP). These are probably not very important under normal circumstances.
In bacteria, aspartate transcarbamylase is the control enzyme. There is only one carbamoyl phosphate synthetase in bacteria since they do not have mitochondria. Carbamoyl phosphate, thus, participates in a branched pathway in these organisms that leads to either pyrimidine nucleotides or arginine.
Interconversion of Nucleotides
The monophosphates are the forms synthesized de novo although the triphosphates are the most commonly used forms. But, of course, the three forms are in equilibrium. There are several enzymes classified as nucleoside monophosphate kinases which catalyze the general reaction:(= represents a reversible reaction)
Base-monophosphate + ATP = Base-diphosphate + ADP
e.g. Adenylate kinase: AMP + ATP = 2 ADP
There is a different enzyme for GMP, one for pyrimidines and also enzymes that recognize the deoxy forms.
Similarly, the diphosphates are converted to the triphosphates by nucleoside diphosphate kinase :
There may be only one nucleoside diphosphate kinase with broad specificity. One can legitimately speak of a pool of nucleotides in equilibrium with each other.
Salvage of Bases
Salvaging of purine and pyrimidine bases is an exceedingly important process for most tissues. There are two distinct pathways possible for salvaging the bases.
The more important of the pathways for salvaging purines uses enzymes called phosphoribosyltransferases (PRT) :
PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotide.:
Base + PRPP = Base-ribose-phosphate (BMP) + PPi
We gave already seen one example of this type of enzyme as a normal part of de novo synthesis of the pyrimidine nucleotides, - O-PRT.
As a salvage process though, we are dealing with purines. There are two enzymes, A-PRT and HG-PRT. A-PRT is not very important because we generate very little adenine. (Remember that the catabolism of adenine nucleotides and nucleosides is through inosine). HG-PRT , though, is exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from free adenine.
HG-PRT is deficient in the disease called Lesch-Nyhan Syndrome , a severe neurological disorder whose most blatant clinical manifestation is an uncontrollable self-mutilation. Lesch-Nyhan patients have very high blood uric acid levels because of an essentially uncontrolled de novo synthesis . (It can be as much as 20 times the normal rate). There is a significant increase in PRPP levels in various cells and an inability to maintain levels of IMP and GMP via salvage pathways. Both of these factors could lead to an increase in the activity of the amidotransferase.
A second type of salvage pathway involves two steps and is the major pathway for the pyrimidines, uracil and thymine.
Base + Ribose 1-phosphate = Nucleoside + Pi (nucleoside phosphorylase)
Nucleoside + ATP - Nucleotide + ADP (nucleoside kinase - irreversible)
There is a uridine phosphorylase and kinase and a deoxythymidine phosphorylase and a thymidine kinase which can salvage some thymine in the presence of dR 1-P.
Formation of Deoxyribonucleotides
De novo synthesis and most of the salvage pathways involve the ribonucleotides. (Exception is the small amount of salvage of thymine indicated above.) Deoxyribonucleotides for DNA synthesis are formed from the ribonucleotide diphosphates (in mammals and E. coli ).
A base diphosphate (BDP) is reduced at the 2' position of the ribose portion using the protein, thioredoxin and the enzyme nucleoside diphosphate reductase . Thioredoxin has two sulfhydryl groups which are oxidized to a disulfide bond during the process. In order to restore the thioredoxin to its reduced for so that it can be reused, thioredoxin reductase and NADPH are required.
This system is very tightly controlled by a variety of allosteric effectors. dATP is a general inhibitor for all substrates and ATP an activator. Each substrate then has a specific positive effector (a BTP or dBTP). The result is a maintenance of an appropriate balance of the deoxynucleotides for DNA synthesis.
Synthesis of dTMP
DNA synthesis also requires dTMP (dTTP). This is not synthesized in the de novo pathway and salvage is not adequate to maintain the necessary amount. dTMP is generated from dUMP using the folate-dependent one-carbon pool.
Since the nucleoside diphosphate reductase is not very active toward UDP, CDP is reduced to dCDP which is converted to dCMP. This is then deaminated to form dUMP. In the presence of 5,10-Methylene tetrahydrofolate and the enzyme thymidylate synthetase , the carbon group is both transferred to the pyrimidine ring and further reduced to a methyl group. The other product is dihydrofolate which is subsequently reduced to the tetrahydrofolate by dihydrofolate reductase.
Thymidylate synthetase is particularly sensitive to availability of the folate one-carbon pool. Some of the cancer chemotherapeutic agents interfere with this process as well as with the steps in purine nucleotide synthesis involving the pool.
Cancer chemotherapeutic agents like methotrexate (4-amino, 10-methyl folic acid) and aminopterin (4-amino, folic acid) are structural analogs of folic acid and inhibit dihydrofolate reductase. This interferes with maintenance of the folate pool and thus of de novo synthesis of purine nucleotides and of dTMP synthesis. Such agents are highly toxic and administered under careful control.
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Etiology and Pathogenesis of Systemic Lupus Erythematosus
Considerable gains in understanding the mechanisms of lupus pathogenesis have established the knowledge base that will underlie future advances in development of targeted therapies to achieve improved patient outcomes. 117,118 The recognition of a central role for innate immune system activation, including the role of type I IFN in lupus pathogenesis, is a major advance of recent years. Rapid progress in characterizing the genetic variants that are associated with a diagnosis of lupus has provided strong evidence that alterations in molecular pathways that regulate genome integrity, nucleic acid degradation , TLR-dependent and -independent innate immune signaling, and lymphocyte activation thresholds and signaling efficiency are important mechanisms that determine disease susceptibility. The products of the immune system, including autoantibodies and their immune complexes, cytokines, and complement components, along with pro-inflammatory mediators and reactive oxygen products released from neutrophils and macrophages, remain key mediators of tissue damage. However, an additional pathogenic role for nucleic acid–containing immune complexes as immune modulators through their important capacity to access and activate endosomal TLRs, as well as insights into regulation of endogenous cytoplasmic nucleic acids, are important new concepts that can guide future therapeutic approaches. Targeting the components of innate immune system activation that affect the TLR pathway along with modulation of pathogenic T cell help for B cell differentiation to autoantibody-producing cells, particularly that mediated by T follicular helper cells, would appear to be a rational therapeutic strategy in light of our current understanding of pathogenic mechanisms.
The references for this chapter can also be found on ExpertConsult.com .
Conclusively, The function of dNTPs in PCR reaction is as important as Taq DNA polymerase .
With the help of Taq , dNTPs bind with the growing DNA strand and expand it. If you don’t want to mess with all this stuff, you can use ready to use PCR kit which is more preferable. Yet, you have to learn the manual method of reagent preparation.
Comment below and let me know if any point is missing from the explanation. Do share your views here.
Quick Notes on Genetic Code | Cell Biology
Living things depend on proteins for exis­tence, the latter produce enzymes necessary for all chemical reactions. Structural infor­mation required to specify the synthesis of any given protein resides in the molecule of DNA which has the spatial configuration of a double helix proposed by Watson and Crick (1953).
The linear sequence of bases in DNA consti­tutes alphabet (hereditary lettering of 4 bases – A, T, C, C) which ‘codes’ for another linear structure, a protein, written in another alphabet of 20 amino acids.
The actual transfer of infor­mation is, however, indirect. DNA is a ‘tem­plate’ for the formation of RNAs, which are incorporated into ribosomes and in turn act as templates for protein synthesis.
All properties of protein, including its secondary and tertiary structure, are ultimately determined by chro­mosomal DNA, and all biological properties are in turn determined by the amino acid sequence of the proteins within an organism, through protein structure and enzyme activity.
The term ‘coding’ implies the relationship between DNA and protein. By coding, the hereditary lettering carried in the four alphabet of DNA is ultimately converted into the protein language composed of twenty letter alphabet of amino acids.
Co-linearity of Gene and Polypep­tide:
In 1958, Crick proposed the hypothesis that DNA determines the sequence of amino acids in a polypeptide. Fundamental to this relationship is that they are both linear in structures, in one case a sequence of nucleotides, in the other case a sequence of amino acids.
By comparing the nucleotide sequence of a gene with the amino acid sequence of a protein, we can determine directly whether the gene and the protein are co-linear or not. A gene of 3N base pairs is required to code for a protein of N amino acids.
The co-linearity of gene and protein was ori­ginally investigated in the tryptophan synthetase gene of E. coli by Yanofsky and his co-workers by utilizing a polypeptide chain A of tryptophan synthetase enzyme. It has been observed that different mutations in the DNA sequence were present in the same order as is observed in the alterations noticed in corresponding amino acid sequence in polypeptide chain A.
The recom­bination distances are relatively similar to the actual distances in the protein, so in this case there is much similarity between the recombina­tion map and the physical map.
For eukaryotic split gene having introns where all base sequences are not translated into amino acid in proteins demonstrates that co-linearity between base sequence of gene and amino acid sequence in protein may be interrupted but not violated.
Properties of Genetic Code:
Code is Triplet:
Researches have been carried out by Ochoa, Kornberg, Nirenberg, Brenner, Crick and others to detect the coding ratio, i.e., the number of units in one system required to specify one unit in the other system. Certainly no one-to-one correspondence can be observed between nucleotides and amino acids.
If each kind of nucleotide specified a single amino acid, only proteins consisting of four amino acids could be constructed. Similarly, the correspondence of an amino acid to two nucleotides would give a larger number of possibilities but still not enough, only = 16.
If a three digit code is employed, however, a total of = 64 kinds of units or codons are established (Fig. 15.1), more than enough to encode twenty amino acids. The surplus forty four triplets were initially thought to be nonsense codons and the remaining twenty as sense codons.
However, later studies have shown that several triplets can code for one amino acid. As such the number of nonsense triplets is very few. Some of the nonsense triplets might also be used as ‘punctuations’, designating the end of a chemical message.
Critical information on the nature of coding units (i.e., the code is in triplets) was gathered from studies of the muta­genic effect on polynucleotide chain (DNA).
Application of mutagen leads to the deletion or duplication of one nucleotide pair or several adjacent pairs. Addition or deletion of one or two bases respectively often causes a drastic effect and the organisms ultimately dies.
The addition or deletion of three bases together, on the other hand, though causing changes in the behaviour of the organism, yet may not necessarily induce a lethal effect and organism may survive with altered mutated tissue.
(i) The direct and exact evidence suppor­ting the triplet code concept was provided by Crick et al. (1961) based on their experiments on a virus, T4 bacteriophage (Fig. 15.2). They found, that the treatment with a chemical called pro-flavin either added or removed a base in its DNA molecule, thus damaging the virus and resulting in an altered or mutant form of the virus.
An addition followed by a deletion of base close by resulted in the restoration of the original virus. This implied that the normal sequences of bases in the DNA molecule had been restored by the second change.
A deletion or insertion completely upsets the reading frame as may be seen from the example of the base sequence GTCCAGACC. Normally the sequence will be read as GTC, CAG, ACC, …, but with the insertion of a new base T between the first and second nucleotides, it yields the sequence GTTCCAGACC … and leads to reading in the groups GTT, CCA, GAC, C …, and specifies wrong amino acids.
A similar con­sequence results from a deletion. Crossing between an addition and deletion will restore the correct reading frame of the sequence except in the region between them. It is easy to see that the combinations of two mutants in the form of two insertions or two deletions will still produce a misplaced reading frame.
Crick (1961) found that three additions or deletions of adjacent nucleotides resulted in the production of the normal virus, due to the restoration of the normal base sequence in DNA.
Thus experiments demonstrating that a combina­tion of three insertions or deletions produced a bacteriophage of perfectly normal appearance and that recombinants containing insertions or deletions in numbers not multiples of three pro­duce only nonfunctional or wrong protein, pro­vided strong evidence that the genetic code operates as a triplet code or that one triplet of nucleotides constitutes a codon.
(ii) The triplet nature of the code was fur­ther confirmed through the research work of Nirenberg and Leder (1965) who found that although little binding of tRNA was possible in the presence of dinucleotide messengers, it occurred preferentially with trinucleotides.
They were able to stimulate binding of different amino acids through different sequences of the same three bases, once again giving credence to the existence of a triplet code.
Code is Non-Overlapping:
In nature, there is always a tendency towards economy. As suggested by Gamow, in his ‘over­lapping’ coding hypothesis, the code is in the form of triplets, but not arranged in a straight chain. It is overlapping in the regions where a particular nucleotide serves in more than one coding unit.
Gamow suggested overlapping code on the basis of two characteristics:
(a) Distance between two bases in a DNA molecule is 3.4A
(b) In a protein molecule also, the distance between two adjacent amino acids is 3.4A.
This can be explained in cases of mono-coding as well as overlapping coding but this is quite impro­bable in a straight chain triplet coding. In the non-overlapping code six nucleotides would code for two amino acids, while in case of over­lapping code up-to four (Fig. 15.3).
In the non- overlapping code each letter Is read only once while in the overlapping code it would be read three times, each time as a part of different words. Mutational changes in one letter would affect only one word in the non-overlapping code while it would affect three words in the overlapping code.
There are evidences of non- overlapping nature of genetic code.
(i) The experimental evidence by Crick (1961) compellingly argued against an over­lapping code and through their research substan­tiated the arguments provided by earlier scien­tists in favour of a non-overlapping code. They started with a messenger of known triplet sequence and used this to synthesize a particular protein.
On adding a nucleotide to it, the parti­cular protein could no longer be synthesized. The result remained unaltered even with the addition of a second necleotide. The proper function of the nucleotide was restored, how­ever, on introduction of a third nucleotide.
A given nucleotide sequence ACTACTAC- TACT bears the codons ACT, ACT, ACT, ACT under the non-overlapping coding systems. An insertion of a nucleotide G between the first C and the first T, under such a system will change the nucleotide sequence to ACGTACTACTACT and codon sequences to ACG, TAG, TAG, TAG, T.
The synthesis of original protein will not take place after the addition of a nucleotide. Instead the altered amino acid chain will be producing an altogether different protein. A second inser­tion of another nucleotide G between the first C and first G of the previously altered nucleotide chain results into a new nucleotide sequence ACGGTACTACTACT and the corresponding codon sequence ACG, GTA, CTA, CTA, CT.
The particular protein still cannot be synthesized. A third nucleotide addition, an insertion of nucleotide G, in the beginning of the nucleotide chain available after the last step causes it to read as GAGGGTACTACTACT and the corresponding codon chain available is GAC, GGT, ACT, ACT, ACT.
The third addition has restored most of the original triplet sequence. The deletion of bases from DNA has the same effect as that of deletion. The third deletion will, however, restores most of the reading frame and allow a sequence of amino acids, differing slightly from its original one. This suggests that the code is non-overlapping.
(ii) Another evidence supporting the exis­tence of a non-overlapping code is provided by the effect of single-site mutations.
A single muta­tion in an overlapping coding system would invariably affect two or more adjacent amino acids in the nucleotide chain. A mutation from the first G to C in the nucleotide sequence ATGATGATG will cause change in one codon only in the case of a non-overlapping code. The original codon sequence of ATG, ATG, ATG will result into a codon sequence ATC, ATG, ATG after single mutation.
However, if the code was an overlapping one, the original codon sequence ATG, TGA, GAT, ATG, TGA, GAT, ATG will change into the codon sequence ATC, TGA, CAT, ATC, TGA, GAT, .ATG. As a result of single mutation, three changes take place. In the codon sequence when the overlapping code is in ope­ration.
Only one change would be expected in case of a non-overlapping code. Since only sin­gle amino acid changes have been observed in the experimental studies of single-site mutation, this evidence reinforces the existence of non-overlapping code.
(iii) Brenner (1957), on the basis of all the published data on the studies of the sequence of amino acids in proteins, concluded that there were no forbidden zones in proteins, and neigh­bouring amino acids were invariably coded by unrelated groups of nucleotides.
It was further established that no specific amino acid will always have the same nearest neighbours and the amino acid sequences appear to be almost completely at random. Such revelations would not have been feasible had the code been of an overlapping nature.
(iv) Yanofsky (1963) provided perhaps the most convincing evidence available that excludes any overlapping code. In his studies of both mutation and recombination through transduc­tion technique, he found that in each protein with a different amino acid at a given position, the amino acids on either side remained unchanged.
Code is Degenerate:
Sometimes three or four triplet codons code for a particular amino acid. Such a genetic code where there are more than one triplet (codon) codes for a single amino acid is known as degen­erate code. Out of possible 64 different codons, 61 codons code for different amino acids.
As there are 20 amino acids, so it is obvious that more than one codon or triplet codes for one amino acid. If each amino acid is coded by a single codon, 44 codons out of 64 will be useless or nonsense codons.
Numerous evidences indicate that the genetic code is degenerate.
(i) If twenty triplets only would have made sense and the remaining forty four remained non­sense, then in a chromosome length mutations could occur only at very limited sites representing one-third of the length and not throughout its entire length.
But the rate of spontaneous muta­tion as well as the results of induced mutation through X-rays has shown that nearly the entire chromosome site is capable of undergoing muta­tion. It is possible if only when the code is degene­rate. However, though the degenerate nature of the code has been established, the presence of high number of repeated sequences may make major segments of chromosomes non-mutable.
(ii) When two bases U and C, in a 3:1 pro­portion are synthesized into in RNA, the possible triplets and their frequency can be mathemati­cally determined :
UUU = 3/4 x 3/4 x 3/4 = 27/64 UUC = 3/4 x 3/4 x 1/4 =9/64 UCU = 3/4 X 1/4 X 3/4 = 9/64 CUU = 1/4 x 3/4 x 3/4 = 9/64 UCC = 3/4 x 1/4 X 1/4 = 3/64 CUC = 1/4 x 3/4 X 1/4 = 3/64 CCU = 1/4 x 1/4 x 3/4 = 3/64 CCC = 1/4 X 1/4 X 1/4 = 1/64.
mRNA of this compo­sition should guide the incorporation of eight amino acids but in fact only four amino acids were actually detected in the protein chain indi­cating the degenerate nature of the code, i.e., some of the codons in this case have directed the incorporation of the same amino acid.
(iii) According to the wobble hypothesis of Crick (1966), the first two bases of the triplet codon pair according to the set rules, i.e., A with U and G with C but the third base having much more freedom of movement than the other two, wobbles and permits more than one type of pair­ing at that position. Thus the wobble hypothesis explains the degeneracy of the code to some extent.
It is sometimes argued that the third base of a code is not very important and that specificity of a codon is particularly determined by the first two bases. It has been shown that the same tRNA can recognise more than one codons differing only at the third posi­tion. This paring is not very stable and is allowed due to wobbling in base pairing at this third posi­tion.
Crick in 1965 proposed a hypothesis called wobble hypothesis to explain this phenomenon. He discovered that if U is present at first position of anticodon, it can pair with either A or G at the third position of codon. Similar is the case with G, found in anticodon, which can pair with either C or U of codon (Table 15.1 A).
The wobble hypothesis visualizes that many codons are able to tolerate mutations at the third base site because of the non-restrictive spatial limitations for the corresponding base in the anti- codon. The third nucleotide in many codons was better tolerated and could be substituted without damage.
The corresponding base in the anticodon would wobble and accommodate. This kind of wobbling allows economy of the number of tRNA molecules since several codons meant for same amino acid are recognized by same tRNA.
Code is Comma-less:
A comma-less code means that no punctua­tion marks are needed between two words. In other words, we can say that after one amino acid is coded, the second amino acid will be automatically coded by the next three letters and no letters are wasted (Fig. 15.4).
However, the code for an entire polypeptide having several amino acids is always terminated by a nonsense codon which servers as full stop in the coding terminology.
If the genetic code functions with commas, a specific nucleotide serves as a punc­tuation mark. Through experiments it has been established that poly-A (AAA) codes for lysine, poly-C (CCC) for proline, and poly-U (UUU) for phenylalanine, which implies that the commas are not made up of A, C and U.
Code is Non-Ambiguous:
Ambiguity denotes that a single codon may code for more than one amino acid. Non- ambiguous means that there is no ambiguity about a particular codon. A particular codon will always code for the same amino acid.
The genetic code is generally non-ambiguous, can be experimentally confirmed using a specific single triplet-ribosome complex which directs the binding of specific tRNA. For example, UUU triplet-ribosome complex directs the binding of phenylalanine-tRNA and AAA triplet-ribosome complex directs the binding of the lysine-tRNA.
In the similar manner, by using the triplets of known sequence, the codons for valine, cysteine, leucine and some other amino acids were determined, thus clearly establishing the non-ambiguous nature of the genetic code under natural physiological conditions.
Code is Universal:
The genetic code is universal. It means that the same codon codes for the same amino acid in all the organisms, from human beings to virus.
Universal nature of genetic code has been experimentally evidenced.
(i) The crucial point in the genetic code is the fitting of tRNA with specific anticodon into the codon of the mRNA.
Thus if mRNA is taken from an eukaryote and tRNA from a prokaryote and protein synthesis could be carried as coded in the mRNA, then it can be proved that code is universal, if mRNA and ribosome are taken from E. coli, and amino acid and tRNA from rat, pro­tein synthesis can be carried out as coded in the mRNA of E. coli. This is true also the other way round.
Von Ehrenstein and Lipmann found that E. coli tRNA to which labeled amino acids were added would form haemoglobin when incubated with the mRNA and ribosomes of rabbit reticulo­cytes.
The precision with which this interspecific attachment occurs was shown by converting cysteine into alanine in amino acid-activated tRNAcys and then observing that this alanine was now inserted into peptide positions ordinari­ly occupied by cysteine, in other words, the anti- codon of the cysteine-tRNA of a bacterial species recognized the cysteine codon of mammalian mRNA in spite of the fact that the tRNA was carrying an alanine amino acid.
(ii) The tRNA from E. coli, Xenopus laevis and guineapig bind to the same trinucleotides as shown by Nirenberg et al., indicates the univer­sality of the code.
(iii) Studies of Merril and co-workers (1971) revealed that a bacterial enzyme X-D-galactose -1 phosphate uridyl transferase which catalyses the metabolism of galactose sugars is produced in human tissue culture cells, previously unable to make it, after infection by a virus carrying the E. coli gal + gene. This provides strong evidence in favour of the universality of the code.
(iv) The correlated nucleotide and amino acid sequences in the overlapping genes of the DNA bacteriophage ф x 174 and in the capsid protein coding gene of RNA bacteriophage MS2 indicates that the genetic code is universal.
(v) Uniformity in amino acid sequence of homologous proteins, e.g., cytochrome c collec­ted from widely divergent species like human, horse, chickens, yeast and bacteria displayed universality of the genetic code.
(vi) Finally genes from human and other organisms have been expressed in E. coli and those from bacteria and other organisms in plants. In each such case, the polypeptide produced by a gene in the new organism was identical with the one it produced in the orga­nism of its origin.
Exceptions of Genetic Code:
A triplet codon demands its own tRNA with a complementary anticodon or a single tRNA responds to both members of a codon pair or to all (or at least some) of the four members of a codon family. Often one tRNA can recognise more than one codon, i.e., codon is degenerate.
This means that the base in the first position of the anticodon must be able to partner alternative bases in the corresponding third position of the codon. In such cases there may be differences in the efficiencies of the alternative recognition reactions (as a general rule, codons that are com­monly used tend to be more efficiently read).
In addition to the constructions of a set of tRNAs able to recognise all the codons, there may be multiple tRNAs that respond to the same codon. The predictions of wobble pairing accord very well with the observed abilities of almost all tRNAs. But there are exceptions in which the codons recognized by a tRNA differ from those predicted by the wobble rules.
Such effects pro­bably result from the influence of neighbouring bases and/or the conformation of the anticodon loop in the overall tertiary structure of the tRNA. Indeed, the importance of the structure of anti­codon loop is inherent in the idea of the wobble hypothesis itself.
Further support for the influ­ence of the surrounding structure is provided by the isolation of occasional mutants in which a change in a base in some other region of the molecule alters the ability of the anticodon to recognize codons.
Another unexpected pairing reaction is pre­sented by the ability of the bacterial initiator, fMet-tRNA ƒmet to recognize both AUG and GUG. This misbehavior involves the third base of the anticodon. Though the genetic code is non-ambiguous, but GUG codes for methionine when used as initiator codon, but it codes for valine if present at the intercalary position, indi­cating its ambiguous nature.
The universality of the genetic code is stri­king, but some exceptions exist. They tend to affect the codons involved in initiation or termi­nation and result from the production (or absence) of tRNAs representing certain codons. Almost all of the changes found in principal genomes affect termination codons.
In the prokaryote Mycoplasma capricolum, UGA is not used for termination, instead codes for tryptophan. In fact, it is the predominant Trp codon, and UGG is used only rarely. Two Trp-tRNA species exist, with the anticodons UCA (reads UCA and UGG) and CCA (reads only UGG).
Some ciliates (unicellular protozoa) read UAA and UAG as glutamine instead of termina­tion signals. Tetrahymena thermophile, one of the ciliates, contains three tRNAglu species. One recognises the usual codons CAA and CAG for glutamine, one recognises both UAA and UAG (according to wobble hypothesis), and the last recognizes only UAG.
We assume that the release factor eRF has a restricted specificity, compared with that of other eukaryotes.
In another ciliate (Euplotes octacarinatus), UGA codes for cysteine. Only UAA is used as a termination codon, and UAG is not found. The change in meaning of UGA might be accom­plished by a modification in the anticodon of tRNAcys to allow it to read UGA with the usual codon UGU and UGC.
The only substitution in coding for amino acids occurs in a yeast (Candida), where CUG means serine instead of leucine (and UAG is used as a sense codon).
All of these changes are sporadic, which is to say that they appear to have occurred indepen­dently in specific lines of evolution. They may be concentrated on termination codons, because these changes do not involve substitution of one amino acid for another. Thus the divergent uses of the termination codons could represent their ‘capture’ for normal coding purposes.
Exceptions to the universal genetic code also occur in the mitochondria from several species.
The earliest change was the employment of uni­versal stop codon UGA to code for tryptophan which is common to all (non-plant) mitochon­dria. It is not likely that UGA coded for trypto­phan in the universal code, but was changed to termination in cytoplasmic translation, because it is a stop codon in bacteria, plant mitochondria and nuclear genomes.
Departures from the universal code, all in non-plant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in Platyhelminthes and echinoderms), UAA (stop) for tyrosine (in Planaria), and AGR (arginine) for serine (in several animal orders and for stop (in vertebrates) [N = A, U, G or C R = A or G) (Table 15.1B).
The mitochondria of plants and protozoans differ in importing and utilizing tRNAs encoded by the nuclear as well as the mitochondrial genome, whereas in animal mitochondria, all the tRNAs are encoded by the organelle.
The small number of tRNAs encoded by the mitochondrial genome highlights an important feature of the mitochondrial genetic system — the use of a slightly different genetic code, which is distinct from the universal code used by both prokaryotic and eukaryotic cells.
Some of these changes make the code simpler, by-replacing two codons that had different meanings with a pair that has a single meaning. Pairs treated like this include UGG and UGA both Trp instead one Trp and one termination) and AUG and AUA (both Met instead of one Met and other lie).
The changes are typically prece­ded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon.
The code reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assign­ment. Changes in release factors also contribute to this revised assignment. Thus the genetic code, formerly thought to be frozen, is now known to be in a state of evolution.
Decipherence of Genetic Code:
It was not possible to say which codon of the possible 64 codons should code for which of the 20 amino acids until the first clue to this problem came when M.W. Nirenberg used in vitro sys­tem for the synthesis of a polypeptide using an artificially synthesized mRNA molecule.
In 1961 Nirenberg and Mathaei characterized the first specific coding sequences, which helped in analysis of genetic code.
Their success on decipherence of code was dependent on two experimental systems:
(i) In vitro (cell free) protein synthesizing system,
(ii) An enzyme, polynucleotide phosphorylase which allowed the synthesis of synthetic mRNAs. These mRNAs served as templates for polypeptide synthesis in the cell free system.
The enzyme polynucleotide phosphorylase functions metabolically in bacteria to degrade RNA, but with high concentrations of ribo­nucleotide diphosphates, the reaction can be ‘forced’ in the opposite direction to synthesize RNA.
Like RNA polymerase it does not require any DNA template, each addition of ribo­nucleotide is random based on the relative concentration of the four ribonucleoside diphos­phates added to the reaction mixtures. The probability of insertion of a specific ribonucleo­tide is proportional to the availability of that molecule, relative to other available ribonucleo­tides.
The cell free system for protein synthesis and the availability of synthetic mRNAs provided a means of deciphering the ribonucleotide compo­sition of various triplets encoding specific amino acids.
Homopolymers Technique (Poly U Experiment):
In their initial experiments, Nirenberg and Mathaei, synthesized RNA homopolymers, each consisting of only one type of ribonucleotide, i.e., the produced mRNA in the in vitro system is either UUUUU …, AAAAA …, CCCCC … or GGGGG … In testing each mRNA, it was very much easy to determine which amino acid was incorporated in the polypeptide chain.
Different amino acids were labelled by using 14 C and tested separately by radioactive counting. In the synthesized RNA using only uracil, there was no other base all along the length of mRNA and the only possible triplet was UUU.
When such a poly-U (RNA) was used in the synthesis of a polypeptide (using all extracts from E. coli, and supplying all the required components of protein synthesizing machinery), only polyphenylalanine was synthesized, meaning that the only amino acid coded was phenylalanine.
It was, therefore, immediately concluded that the input UUU coded for the amino acid phenylalanine. Subsequently, poly A gave polylysine and poly C gave poly-proline. Therefore, UUU was assigned to phenylalanine, AAA to lysine and CCC to pro­line. But the poly G did not serve as template as it gets folded backs on itself, for this assignment other method had been followed.
Heteropolymers (Random): Mixed Copolymers Technique:
The study of polynucleotides were further extended with copolymers as synthetic messen­gers containing two or more bases in definite proportion in cell free system. These randomly synthesized polynucleotides resulted in direct incorporation of amino acids into protein in a manner which indicated that a number of different code words are involved in the binding of different amino acids.
In cell free culture, with these synthetic polyribonucleotide’s, the different amino acids incorporated in a messenger could be clearly correlated with the expected variations in the frequency of different triplets in the synthetic copolymers. Thus this experiment showed the way of deriving nucleotide composition of triplets for each of the amino acids.
Nirenberg, Mathaei and Ochoa did their experiments using the RNA heteropolymers in this technique two or more different ribonucleoside diphosphates were added in combination to form the artificial message. The frequency of a particular triplet codon on the synthetic mRNA depended on the relative proportion of ribo­nucleotide addition in the cell free system.
The percentage of incorporation of particular amino acid in the polypeptide chain could be used for prediction against a particular triplet codon.
For example, in a system A and C are added in a ratio of 1 A: 5C. Now, the insertion of a ribonu­cleotide at any position along the RNA molecule during its synthesis is determined by the ratio of A:C. Therefore, there is a 1/6 possibility for an A and a 5/6 chance for a C to occupy each position.
On this basis, we can calculate the frequency of any given triplet appearing in the message. For AAA, frequency is (1/6) 3 or 0.4%. For AAC, ACA and CAA, the frequencies are identical (1/6) 3 x 5/6 or 2.3%, all three together it is 6.9%. In the same way 1A:2C is calculated which is 1/6 x (5/6) 2 or 11.6% or all together 34.8%, whereas CCC is (5/6)3 or 57.9% of the triplets.
Now by examining the percentage of any given amino acid incorporated into the protein synthesized under the direction of this message, it is possible to propose probable base composi­tion. As because proline appears 69%, it can be deduced that proline is likely to be coded by CCC (57.9%) and also by one of the triplet code 1A : 2C variety (11.6%), i.e., 57.9 + 11.6.
Histidine incorporation percentage is 14% which is probably coded by one 1A:2C category and another 1C:2A category (11.6+2.3)%. Threonine shows 12% incorporation, i.e., likely to be coded by one 1A:2C category. Asparagine and glutamine appear to be coded by one of the 1C:2A triplets and lysine appears to be coded by AAA.
Using as many as all four ribonucleotides to construct this kind of random heteropolymers of synthetic mRNA, the composition of triplet code words corresponding to all 20 amino acids could be determined (Table 15.2).
Heteropolymers (Ordered): Repea­ting Copolymers Technique:
In early 1960s H.G. Khorana could chemi­cally synthesize long RNA molecule consisting of short sequences repeated many times. The short sequences were of di-, tri- or tetra-nucleotides, which were replicated many a times and finally joined enzymatically to form the long polynu­cleotides.
The dinucleotide repeats will be trans­lated for two different amino acids trinucleotide repeats will be converted into 3 potential triplets, depending on the point at which initiation occurs and a tetra-nucleotide creates four repea­ting triplets.
When these synthetic mRNAs were added to a cell free system and amino acid incorporation is matched, the conclusions can be drawn from the composition assignment and triplet binding, and specific assignments were possible.
When the repeating dinucleotide sequence is UCUCUCUC…, it produces the triplets UCU and CUC — they can incorporate leucine and serine into the polypeptide. When the repeating trinucleotide sequence is UUCUUCUUC…, the possible triplets are of three kinds: UUC, UCU and CUU depending on the initiation point and they can incorporate phenylalanine, serine and leucine.
From the above two results it can be concluded that UCU and CUC encode for serine and leucine and also either UUC or CUU encodes for serine or leucine, while the other encodes for phenylala­nine. Further, when the tetra-nucleotide sequence UUAC is repeated then it produces the UUA, UAC, ACU and CUU.
Here the incorporated amino acids are leucine, threonine and tyrosine. In the above two cases, the common code is CUU and common amino acid incorporated is leucine, so it can be concluded that CUU encodes for leucine.
Now from these experiments logically it can be determined that UCU encodes for serine and the rest UUC encodes for phenylalanine and also the CUC encodes for leucine (Table 15.3).
Like this way, by logical interpretations, Khorana reaffirmed triplets that were already deciphered and filled in gaps left from other approaches (Table 15.4).
Triplet Binding Technique:
Nirenberg and Leder in 1964 found that if a synthetic tri-nucleotide for a known sequence is used with ribosome and a particular aminoacyl- tkNA, these will form a complex provided that the used codon codes for the amino acid attached to the given aminoacyl-tRNA.
In order to work out the code for all 20 amino acids, all the possible 64 triplets had to be tried in cell free culture.
In the experiment, 20 samples of the mixture of all 20 amino acids were taken and in each sample, one amino acid was made radioactive in such a manner that each and every amino acid is radioactive in one sample or the other, and no two samples have same radioactive amino acid. For instance, in one set valine has been labelled and the rest 19 remained unlabelled.
Similarly, in another set lysine was labelled and the rest 19 remained un-labelled. Then the tRNAs and ribosomes are mixed with each of these samples and the same codon is used for all sets. When the mixture is poured on the nitro­cellulose membrane, radioactivity on membrane will be observed only when the radioactive amino acid is taking part in the formation of complex.
Since in each sample the radioactive amino acid is known, it would be possible to detect the amino acid coded by a given codon by the presence of radioactivity on the membrane. Such a treatment was given to all 64 synthetic codons, and their respective amino acids were identified.
The base sequence in mRNA and the resul­ting amino acid sequence in protein reveals the code for each amino acid. All the 64 codons, along with their amino acids, are represented in Table 15.5.
An examination of the code table reveals the following characteristics:
i. Each codon consists of three nucleo­tides, i.e., the code is triplet. 61 codons represent 20 amino acids. Three represent (UAA, UAG, UGA) punctuation marks for termination of pro­tein synthesis.
ii. Almost all amino acids are coded by more than one codon, except methionine and tryptophan which have only one codon. Phenylalanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic add and cysteine are the nine amino acids which are represented by two codons each. Three amino acids, i.e., arginine, serine and leucine have Six codons each. The table indicates the degeneracy of the genetic, code.
iii. If an amino acid has more than one codon, the first two nucleotides are identical and the third nucleotide can be either cytosine or uracil. Adenine and guanine are also similarly interchangeable at the third position. For example, UUU and UUC, both code for phenylalanine, and UCU, UCC, UGA and UCG code for serine.
However, there are some exceptions to the equi­valence rule of the first two nucleotides, as AGU and AGC also code for serine apart from UCU, UCC, UCA and UCG.
Similarly, the amino acid leucine is also coded- by six codons, i.e., UUA, UUG, CUU, CUC, CUA and CUG.
The frequent interchange of cytosine and uracil or guanine and adenine suggests that great variations can occur in AT/GC ratio in certain organisms without affecting large changes in the relative proportions of amino acids present in them, as for almost every amino acid there is one codon that carries G or C and another that carries A or U as its third nucleotide.
The two organisms carrying the same protein sequence information in their DNA, by selecting one or the other kind of synonym codon, can show different AT/GC ratios.
iv. The genetic code has a definite structure in the sense that the synonyms for the same amino acid are not randomly dispersed over the table but are usually found together. The only exceptions are the codons, six each for arginine, serine, and leucine, which are spread over the table.
v. Multiple codons for an amino acid show in general the similarity in first two nucleotides and it is the third nucleotide which varies.
AUG is the initiation codon, i.e., the polypeptide chain starts with methionine. This amino acid is the formulated form of methionine. The initiation codon binds to fmet-tRNA having an anticodon 3′ UAC 5′ which is identical to that of met-tRNA, i.e., both met- tRNA and fmet-tRNA are coded by AUG but the signal for the starting amino acid is much more complex than the signal for all other amino acids.
According to Stent, there exist two separable species of tRNA capable of accepting methionine. Methionine of only one of these is concerned into formyl methionine by the action of the special formulation enzyme. The other or ordinary met- tRNA incorporates methionine into the interior of the growing polypeptide chain and responds to the codon AUG only.
Formyl-met-tRNA initiates the polypeptide chain and responds to GUG (valine codon) also. The GUG while present at the initiation point, codes for methionine whereas in the intercalary position, it codes for valine. The anticodon of this species of tRNA seems to be per­missive with respect to the first nucleotide base of the codon and selective with respect to the second and third nucleotide bases.
UAA, UAG and UGA are the chain termination codons. They do not code for any of the amino acids but serve as stop codon. These codons do not have any tRNA but are read by specific proteins called release fac­tors. These codons are also called nonsense codons.
A mutation from a sense to nonsense codon in the middle of a genetic message results in the release of immature or incomplete polypeptides which do not have any biological activity. Nonsense mutations can be induced by mutagens. UAG was formerly known as amber, UAA as ochre and UGA as opal.
What are the differences between DNA nucleotides and RNA nucleotides?
How is the information stored within the base sequence of DNA used to determine a cell&rsquos properties?
How do complementary base pairs contribute to intramolecular base pairing within an RNA molecule?
If an antisense RNA has the sequence 5ʹAUUCGAAUGC3ʹ, what is the sequence of the mRNA to which it will bind? Be sure to label the 5ʹ and 3ʹ ends of the molecule you draw.
Why does double-stranded RNA (dsRNA) stimulate RNA interference?
Nucleotide Structure: The following image from wikipedia’s image gallery shows the basic structure of the nucleotide and the five nitrogenous bases.
The central component of all nucleotides will be a pentose sugar (5-carbon sugar). We will either see ribose or 2’deoxyribose as the sugar (the second carbon has one less oxygen than ribose). Off of the 5′ carbon of the sugar, you will find a phosphate group attached, while on the 1′ carbon, you will find a nitrogenous base. [NOTE: remember the numbering of carbon atoms in carbohydrates from yesterday? Do you see why the numbering is important?]
There are five nitrogenous bases, divided into two categories: Purines and Pyrimidines. Notice that the purines are a composite of two ring structures, while the pyrimidines are a single ring structure. When you take organic chemistry and biochemistry, the importance and complexity of these ring structures will be further discussed. At present, just become aware of their respective shapes and sizes (and inclusion of nitrogen ).
As with amino acids, the nucleotide contains a functional group: the nitrogenous base. Just like the side chain in an amino acid, the nitrogenous base will play an important part in the function of this biomolecule. The Sugar-Phosphate then becomes the backbone of the molecule (line the Amino-Chiral Carbon-Carboxyl of an amino acid). We will in later weeks that the sugar-phosphates of nucleotides will create the strands of DNA and RNA. The nitrogenous bases then playing an information role.
NOTE: RNA nucleotides (ATP, GTP, CTP & UTP) will be the nucleotide form most often encountered in the cell. DNA nucleotides (dATP, dGTP, dCTP, and dTTP) will only be found during DNA replication.
The nucleic acids are referred to as informational biomolecules (biopolymers). This is because the sequence of nucleotides carries information on how to build RNA and Proteins. One of the central foundations of genetics (i.e., how it all works), is base complementarity . Here we are looking at the interactions between purines and pyrimidines in DNA:
A links with T through 2 hydrogen bonds.
G links with C through 3 hydrogen bonds.
A to T G to C
U has the binding properties of T, but is only found in RNA.
T is never found in RNA, only DNA.
NOTE: base complementarity is a critical concept to remember. All genetic processes rely on base complementarity!
When we get to genetics, we will be talking about the directionality of the nucleic acids. For example, we will talk about DNA being built from the 5′ to 3′. This is in reference to the carbon atoms in the ribose or deoxyribose. The 5′ holds a phosphate, while the 3′ holds an open -OH (hydroxyl) group. This concept of directionality is critical, and you are warned to learn how it works, and what the terms represent.
As with all biopolymers, monomers are added together through dehydration synthesis, and separation is through hydrolysis. When synthesis occurs, the 5′ phosphate links to the 3′ -OH, forming a phosphodiester bond .
DNTP POOL ASYMMETRIES AS MUTAGENIC DETERMINANTS
As shown through many in vitro studies (reviewed in ref 1), the ability of a mismatched nucleotide to force a replication error, whether through misinsertion or a next-nucleotide effect, is clearly a function of the concentrations of the correct and incorrect dNTPs. Therefore, over evolutionary time we might expect the DNA base composition of an organism to reflect the composition of its dNTP pool at replication sites. In fact, in nearly all organisms analyzed, dNTP pools are highly asymmetric. The most striking general feature is underrepresentation of dGTP, which usually comprises just 5 to 10% of the total dNTP pool (31). For example, we estimated the concentrations of the four dNTPs in synchronized S-phase HeLa cells to be dATP, 60 µM dTTP, 60 µM dCTP, 30 µM dGTP, 10 µM, with dGTP making upjust 6% of the total (32). Although estimates of this type do not take into account possible compartmentation effects, they do provide a useful first approximation.
Genetic consequences of natural dNTP pool asymmetry
It is generally thought that cells are organized to maximize DNA replication accuracy, because spontaneous mutation rates are sometimes lower than expected from the fidelity of replication complexes, even when mismatch repair is taken into account (33). However, there is reason to speculate that cells and organisms strike a balance between replication velocity and accuracy, allowing rapid reproduction while at the same time allowing cells to respond to stress conditions, and ultimately permitting desirable evolutionary variation (34). Might dNTP pool asymmetries contribute by increasing in vivo replication error rates relative to what would be seen if the pools were balanced? In our laboratory, Stella Martomo (35) approached this question by running replication reactions in vitro in the presence of dNTPs either at their estimated in vivo concentrations or equimolar. She found that a 3- to 4-fold underrepresentation of dGTP, comparable to that seen in cells, was not strongly mutagenic. However, if dGTP was increased she found the error rate to increase in direct proportion to the concentration of the nucleotide in excess. This latter result probably deserves further investigation with in vitro systems. For example, Darè et al. (36) used dCMP deaminase deficiency and addition of specific nucleosides to artificially boost pools of single dNTPs in V79 hamster cells, and they reported that dGTP accumulation in vivo is more strongly mutagenic than that of either dCTP or dTTP, a finding without an obvious explanation. Like Darè et al., we have observed a dCMP deaminase deficiency, which causes a specific accumulation of dCTP, to be nonmutagenic (unpublished results).
The limited data available, as just discussed, suggest that natural dNTP pool asymmetries are not strongly mutagenic, at least for nuclear DNA. However, the absolute values of dNTP pools may contribute to mutagenesis, even when those pools are balanced. A review of the literature, plus new data, revealed that mammalian cell lines that have undergone oncogenic transformation contain dNTP pools 3- to 4-fold larger than those of normal diploid cells (35). When Martomo followed DNA replication in cell-free extracts programmed with dNTPs at transformed or normal diploid levels, she found that at normal diploid levels the error frequency was indistinguishable from background levels, whereas at the higher levels characteristic of transformed cells, the error frequency exceeded background values by 2- to 5-fold. This finding suggested that balanced accumulation of dNTPs could be mutagenic. Linda Wheeler tested this in E. coli by expressing recombinant ribonucleotide reductase. As shown in Table 1, this treatment, which caused the dNTP pools to expand by some 3- to 5-fold, caused an increase in the spontaneous mutation frequency out of proportion to the pool accumulation—up to 40-fold (7). We propose that this results from decreased proofreading efficiency at high dNTP levels, resulting from enhanced DNA chain extension from a mismatched 3′ terminal nt. From kinetic properties of replicative DNA polymerases, it seems clear that the enzyme is saturated with dNTPs at physiological levels for DNA chain extension from a correctly matched 3′ terminus, but the rate of extension from a mismatch is far below saturation published values for different polymerases give KM values for mismatch extension in the millimolar range (7). Thus, as dNTP levels increase above normal values, the rate of normal elongation will not increase, but mismatch extension will increase, possibly in direct proportion to dNTP concentrations, with consequent inhibition of proofreading. These relationships are summarized in Fig. 2.