Wobble rules. Types of mutations. Base substitutions. Just as a reminder, there are two types of base substitutions. The same class of nucleotide remains. Examples are A substituting for G or C substituting for T.
Over evolutionary time, the rate of accumulation of transitions exceeds the rate of accumulation of transversions. Effect of mutations on the mRNA. Depending on the particular replacement, it may or may not have a detectable phenotypic consequence. Some replacements, e. Other replacements, such as valine for a glutamate at a site that causes hemoglobin to polymerize in the deoxygenated state, cause significant pathology sickle cell anemia in this example.
They almost always have serious phenotypic consequences. Not all base subsitutions alter the encoded amino acids. However, there are several exceptions to this rule.
This is one of the strongest supporting arguments in favor of model of neutral evolution, or evolutionary drift, as a principle cause of the substitutions seen in natural populations.
The template strand of a sample of double-helical DNA contains the sequence:. Will the resulting amino acid sequence be the same as in b? Explain the biological significance of your answer. In sickle-cell hemoglobin there is a Val residue at position 6 of the b -globin chain, instead of the Glu residue found in this position in normal hemoglobin A. Can you predict what change took place in the DNA codon for glutamate to account for its replacement by valine?
What is the sequence of the original codon for Lys? Deduce the sequence of the wild-type codon in each instance. What is the codon for Gln? What is the codon for Leu? Design a DNA probe that would allow you to identify the gene for a protein with the following amino-terminal amino acid sequence. The probe should be 18 to 20 nucleotides long, a size that provides adequate specificity if there is sufficient homology between the probe and the gene.
While the rest of the crew tries to figure out if the fungus is friend or foe and gets all the camera time , you are assigned to determine its genetic code. With the technologies of two centuries from now, you immediately discover that its proteins are composed of only eight amino acids, which we will call simply amino acids 1, 2, 3, 4, 5, 6, 7, and 8. Its genetic material is a nucleic acid containing only three nucleotides, called K, N and D, which are not found in earthly nucleic acids.
The results of frameshift mutations confirm your suspicion that the smallest possible coding unit is in fact used in this fungus. Insertions of a single nucleotide or three nucleotides into a gene cause a complete loss of function, but insertions or deletions of two nucleotides have little effect on the encoded protein.
You make synthetic polymers of the nucleotides K, N and D and use them to program protein synthesis. The amino acids incorporated into protein directed by each of the polynucleotide templates is shown below. Assume that the templates are read from left to right. Template Amino acid s incorporated. Lieutenant Data tells you that is all you need to figure out the code, but just to check yourself, you examine some mutants of the fungus and discover that a single nucleotide change in a codon for amino acid 6 can convert it to a codon for amino acid 5.
Also, a single nucleotide change in a codon for amino acid 8 can convert it to a codon for amino acid 7. Please report your results on the genetic code used in the fungus from Planet Claire.
Amino acid Codon s. Show both the initial codon and the mutated codon. Size of a codon : 3 nucleotides 1. Experiments to decipher the code 1. Wheat germ extracts c. Bacterial extracts 2. Oligonucleotide synthesis, deprotection, and quality control were carried out as previously described 59 , The synthesis of the benzimidazole nucleotide will be published elsewhere.
Product containing fractions were applied to a C18 SepPak catridge Waters to remove eluent buffer salts. The resolved proteins were blotted to 0. As a secondary antibody, a goat anti-mouse HRP-conjugated antibody Dako, P was used in a dilution. Uncropped western blot scans are depicted in Supplementary Fig.
Database search was performed using ProteomeDiscoverer Version 2. The following settings were applied: Enzyme for protein cleavage was trypsin; two missed cleavages were allowed.
Fixed modification was carbamidomethylcysteine; variable modifications were N-terminal protein acetylation and methionine oxidation. Precursor mass tolerance was set to 10 ppm; fragment mass tolerance was 20 mmu. Oligonucleotide samples were lyophilized to dryness, dissolved in melting buffer and degassed, and a layer of silicon oil was placed on the surface of the solution to avoid evaporation. Initiation complexes were diluted in buffer A to give a range of concentrations 0. In turn, EF—Tu ternary complexes were formed with 0.
Ternary complex reactions were then placed on ice. All subsequent steps were performed with a multichannel pipette. All binding experiments were repeated more than three times. The equilibrium dissociation constant K D was determined by fitting the binding data to a one-site binding hyperbolic equation GraphPad Prism 7.
Further information on research design is available in the Nature Research Reporting Summary linked to this article. All other data supporting the findings of this study are available within this article and in the Supplementary Information or from the corresponding author upon reasonable request. A reporting summary for this article is available as a Supplementary Information file. Leontis, N. The non-Watson-Crick base pairs and their associated isostericity matrices.
Nucleic Acids Res. Moore, P. Structural motifs in RNA. Annu Rev. Watson, J. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature , — Nissen, P. The structural basis of ribosome activity in peptide bond synthesis.
Science , — Guerrier-Takada, C. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35 , — Kruger, K.
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Crick, F. Codon—anticodon pairing: the wobble hypothesis. Structural insights into translational fidelity. Grosjean, H. An integrated, structure- and energy-based view of the genetic code. Wohlgemuth, I. Evolutionary optimization of speed and accuracy of decoding on the ribosome. B Biol. Yusupova, G. The path of messenger RNA through the ribosome. Cell , — Boccaletto, P. Article Google Scholar. Schimmel, P. Cell Biol. Agris, P. The importance of being modified: the role of RNA modifications in translational fidelity.
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Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function. RNA Biol.
Hoernes T. Translating the Epitranscriptome. Wiley Interdiscip. RNA 8 , doi: Hoernes, T. Davalos, V. SnapShot: messenger RNA modifications. Cell , — e Paul, M. EMBO J. Nishikura, K. Krepl, M. Effect of guanine to inosine substitution on stability of canonical DNA and RNA duplexes: molecular dynamics thermodynamics integration study. B , — Manickam, N. Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength.
Ledoux, S. Different aa-tRNAs are selected uniformly on the ribosome. Zhang, Y. A semi-synthetic organism that stores and retrieves increased genetic information. Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.
Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release. USA , E—E Shimizu, Y. Cell-free translation reconstituted with purified components. This shows that tRNA duplications are influenced by existing mutational bias. We presume that the reason the GC skew exists in the low-expression genes is because of context-dependent mutation.
We will not pursue this point here, although we have discussed it in detail in the case of codon usage in mitochondrial genes Jia and Higgs Context-dependent effects in codon frequencies in prokaryotic and eukaryotic genomes have also been found by Moura et al.
There are many cases where the wobble base of the tRNA is modified in a way that influences anticodon—codon pairing Curran ; Agris , , Grosjean et al. In this section, we consider the way that modified bases might influence the interpretation of the codon preference data above.
We presume that this modification occurs in the majority of the bacteria in our sample. However, a detailed study of Mycoplasma capricolum Andachi et al. In Escherichia coli , Urbonavicius et al. However, Meier et al. Also Morris et al.
It seems likely that the reason for the presence of the Q modification is to increase the efficiency of pairing with the U codon. However, our observation is that the C codon is still preferred, even when Q is present; therefore, we conclude that the Q modification does not change the direction of the codon preference.
Another possible function of Q is that it may reduce pairing with the A codon, which might occur if the wobble-G were unmodified. Loss of the Q modification and subsequent GA pairing is relevant with respect to the origin of several variant genetic codes in mitochondrial genomes Yokobori et al.
One of the most surprising results from the codon usage analysis is that the U codon is often preferred in four-codon families when wobble-U tRNAs are the only ones table 2 or the most frequent ones table 3.
This shows that there is an unexpectedly high efficiency of pairing of the UU combination, with a rate b UU that is similar to b UA and higher than b UG. Thus, the codon usage data show that wobble-U tRNAs behave differently in two- and four-codon families.
Base modifications seem to be an important part of the explanation for this. We will denote this class of mutations as xo 5 U. In tRNAs for two-codon families, the U base usually has a 5-methylaminomethyl modification and often a 2-thio modification as well. Examples occurring in bacteria are 5-methylaminomethyl uridine mnm 5 U , 5-carboxymethylaminomethyl uridine cmnm 5 U , 5-methylaminomethyl 2-thiouridine mnm 5 s 2 U , and 5-carboxymethylaminomethyl 2-thiouridine cmnm 5 s 2 U.
We denote this class as xm 5 U. Several cases of xo 5 U modifications have been studied experimentally Lustig et al. The main conclusion from these examples is that the xo 5 U modification enhances the ability of the tRNA to pair with all four codons. This shows that an unmodified U can pair with all four codons, at least to some extent. In contrast, experiments show that the xm 5 U modification restricts pairing to only A and G codons, as is necessary to prevent mistranslation in two-codon families.
However, Ashraf et al. It appears to be significant that the xm 5 U modifications occur even in Mycoplasma Andachi et al. One reason why the two types of modification function in different ways is because they have different effects on the 3D configurations of the ribose.
Yokoyama et al. This is consistent with our observation from the codon usage data that UU pairing is fast, and U codons are often preferred when the xo 5 U modification is present.
Table 2 shows that the C codon is the least preferred in cases where only wobble-U tRNAs are present; therefore, we conclude that UC pairing is possible but is still weak, even in the presence of the xo 5 U modification. It is presumed that C only pairs with G codons.
This is essential for Met and Trp, which have only one codon, but in two- and four-codon families, wobble-C tRNAs are usually an optional extra because the wobble-U tRNA can pair fairly well with the G codon. The U codon is preferred in almost all these cases.
Also, the A codon decreases in frequency in high-expression genes, which is consistent with there being weak interaction between I and A. It is not clear why an unmodified A is rare at the wobble position. Boren et al.
They speculated that wobble-A bases are generally avoided because A would be indiscriminate. This argument makes sense in split codon boxes but does not apply in four-codon families.
Also, if A pairs well with four codons, there must be a reason why it is necessary to modify it to I. Presumably, the I modification must speedup translation of at least the U and C codons relative to the unmodified A, but the reason for avoidance of the unmodified A still seems rather unclear.
We have focused on modifications at the wobble position tRNA position 34 because these have a direct interaction with the third codon position. However, base modifications in other positions are also significant in terms of translation and possibly codon usage. In particular, modifications often occur at position 37 the base that follows the anticodon.
Removal of these modifications has been shown to have a detrimental effect on either speed or accuracy of translation in some cases Yarian et al. Base modifications at positions 34 and 37 have also been found to be important for proper translocation of a tRNA from the A site to the P site in the ribosome Phelps et al.
Finally in this section, we note that tRNAs with the same wobble position base may differ in their ability to pair with alternative codons because of structural differences that have nothing to do with base modifications. Lehmann and Libchaber argued that a single tRNA can pair with four codons when codon—anticodon interactions are strong but not when they are weak. For this reason, weakly interacting codon boxes can be split between two amino acids, whereas strongly interacting boxes must remain as four-codon families.
This argument is interesting in the context of the evolution of the genetic code Lehmann and Libchaber ; Higgs , and in the present context, it provides another explanation of why the wobble-U tRNAs can translate all four codons in four-codon families but not in two-codon families.
The model of translational selection that we have used to interpret the codon usage data above is based on the assumption that translational speed is the key factor. However, it is also possible that translational accuracy plays a role, that is, the preferred codons are those for which the error rate is smallest rather than those that are most rapidly translated. Here, we will discuss both possible causes of selection, and we will argue that there are important aspects of these data that can be most easily explained in terms of selection for speed, although it is quite possible that selection for accuracy is operating at the same time.
First, we note that there is experimental evidence that there is significant difference in translation speeds between synonymous codons. Curran and Yarus and Sorensen and Pedersen found that codons that were preferred in E. It is also known that insertion of blocks of slow codons into a sequence has a significant effect on protein production rate Mitarai et al. More recent experiments have attempted to measure the rates of the different steps involved in the translation cycle for each codon Rodnina and Wintermeyer ; Blanchard et al.
Also, different groups use different kinetic schemes, as pointed out by Ninio , so there is not yet complete agreement on what the underlying steps are. We wish to emphasize that selection on codon bias shows up in the simplest possible case where there is only one tRNA that pairs with two synonymous codons. Thus, if it is speed that is under selection, there must be a difference in the rate at which the ribosome recognizes and processes the tRNA that depends on the details of the codon—anticodon interaction.
Accuracy-based arguments assume that mistranslation has a cost because some mistranslated proteins are nonfunctional or that they misfold more often Drummond and Wilke Selection for accuracy can explain observed differences in codon usage between conserved and variable sites Akashi ; Stoletzki and Eyre-Walker seen in some species, which we would not expect from selection for speed alone.
If accuracy is the key factor, then in order to understand this fully, it will be necessary to measure the mispairing rates of each tRNA with all the non-cognate codons as well as the correct pairing rates with the cognate codons. The relative accuracy of synonymous codons has been measured in a few cases Precup and Parker ; Kramer and Farabaugh , although there is no systematic study of relative accuracy that covers all codons in a given organism.
An interesting special case related to selection against inaccurate codons is the elimination of ambiguously translated codons during periods of codon reassignment, as has been observed with Candida Butler et al.
Despite these caveats regarding the possible relevance of accuracy as well as speed, there are two aspects of the data that point to the fundamental importance of translational speed.
First, it has been shown by Rocha and in our previous paper Higgs and Ran that codon bias is higher in bacteria with faster growth rates. This is a natural expectation if speed is important—bacteria living in a niche where rapid cell division is advantageous need to adapt to optimize their rate of protein synthesis. It is difficult to see why this correlation should occur if selection were solely due to accuracy.
Second, it is found that there are more duplicate tRNA genes in bacteria that are rapidly multiplying and in species where the codon bias is strong Rocha ; Higgs and Ran Our theory predicts in which circumstances duplications are favored by selection for translational speed Higgs and Ran In contrast, it is not clear that selection for accuracy alone will favor gene duplications. Duplicating one tRNA should increase the rate of translation of its cognate codons and hence also increase their accuracy.
However, it will also increase the rate at which this tRNA mispairs with non-cognate codons. It is not clear which of these is more important.
Furthermore, if we make a general duplication of all genes, this will increase all the correct pairing rates and mispairing rates proportionately, so there should be no change in accuracy but a large increase in speed of all codons.
We emphasize that the above arguments apply only to bacteria, and it may well be that speed is less relevant in multicellular organisms than in bacteria. Several examples where arguments for translational accuracy have been made are multicellular eukaryotes Drummond and Wilke However, eukaryotes tend to have large numbers of tRNA genes, and the number of copies of each type of tRNA is correlated with the codon frequencies, as has been shown, for example, in Caenorhabditis elegans Duret and humans Lavner and Kotlar Therefore, tRNA copy number and codon frequencies are also coevolving in eukaryotes, and this means that there is still a fundamental role for speed and efficiency even in multicellular organisms.
Our interpretation of the observed variation in the strength of codon bias among species is that the selection strength s 0 in eq. However, the effective population size, N e , also influences codon frequencies eqs. It is therefore possible that the species with high codon bias correspond to those with the highest N e. We do not have estimates of N e for all species in this data set but Lynch and Conery determined the product N e u for several species where u is the mutation rate , including nine of the bacteria in our data set.
We found no correlation between the strength of codon bias in our data and N e u for these species, whereas codon bias was found to be correlated with growth rate in the same nine species as in the full data set. On the other hand, in prokaryotic organisms, ribosomes can attach to mRNA while it is still being transcribed. In all types of cells, the ribosome is composed of two subunits: the large 50S subunit and the small 30S subunit S, for svedberg unit, is a measure of sedimentation velocity and, therefore, mass.
Each subunit exists separately in the cytoplasm, but the two join together on the mRNA molecule. The tRNA molecules are adaptor molecules—they have one end that can read the triplet code in the mRNA through complementary base-pairing, and another end that attaches to a specific amino acid Chapeville et al. The idea that tRNA was an adaptor molecule was first proposed by Francis Crick, co-discoverer of DNA structure, who did much of the key work in deciphering the genetic code Crick, The rRNA catalyzes the attachment of each new amino acid to the growing chain.
Interestingly, not all regions of an mRNA molecule correspond to particular amino acids. In particular, there is an area near the 5' end of the molecule that is known as the untranslated region UTR or leader sequence.
This portion of mRNA is located between the first nucleotide that is transcribed and the start codon AUG of the coding region, and it does not affect the sequence of amino acids in a protein Figure 3. So, what is the purpose of the UTR? It turns out that the leader sequence is important because it contains a ribosome-binding site.
A similar site in vertebrates was characterized by Marilyn Kozak and is thus known as the Kozak box. If the leader is long, it may contain regulatory sequences, including binding sites for proteins, that can affect the stability of the mRNA or the efficiency of its translation.
Figure 4: The translation initiation complex. When translation begins, the small subunit of the ribosome and an initiator tRNA molecule assemble on the mRNA transcript.
The small subunit of the ribosome has three binding sites: an amino acid site A , a polypeptide site P , and an exit site E. Here, the initiator tRNA molecule is shown binding after the small ribosomal subunit has assembled on the mRNA; the order in which this occurs is unique to prokaryotic cells.
In eukaryotes, the free initiator tRNA first binds the small ribosomal subunit to form a complex. Figure Detail Although methionine Met is the first amino acid incorporated into any new protein, it is not always the first amino acid in mature proteins—in many proteins, methionine is removed after translation.
In fact, if a large number of proteins are sequenced and compared with their known gene sequences, methionine or formylmethionine occurs at the N-terminus of all of them.
However, not all amino acids are equally likely to occur second in the chain, and the second amino acid influences whether the initial methionine is enzymatically removed. For example, many proteins begin with methionine followed by alanine.
In both prokaryotes and eukaryotes, these proteins have the methionine removed, so that alanine becomes the N-terminal amino acid Table 1. However, if the second amino acid is lysine, which is also frequently the case, methionine is not removed at least in the sample proteins that have been studied thus far. These proteins therefore begin with methionine followed by lysine Flinta et al.
Table 1 shows the N-terminal sequences of proteins in prokaryotes and eukaryotes, based on a sample of prokaryotic and eukaryotic proteins Flinta et al. In the table, M represents methionine, A represents alanine, K represents lysine, S represents serine, and T represents threonine.
Once the initiation complex is formed on the mRNA, the large ribosomal subunit binds to this complex, which causes the release of IFs initiation factors. The large subunit of the ribosome has three sites at which tRNA molecules can bind. The A amino acid site is the location at which the aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that correct amino acid is added to the growing polypeptide chain.
The P polypeptide site is the location at which the amino acid is transferred from its tRNA to the growing polypeptide chain. Finally, the E exit site is the location at which the "empty" tRNA sits before being released back into the cytoplasm to bind another amino acid and repeat the process. The ribosome is thus ready to bind the second aminoacyl-tRNA at the A site, which will be joined to the initiator methionine by the first peptide bond Figure 5.
Figure 5: The large ribosomal subunit binds to the small ribosomal subunit to complete the initiation complex. The initiator tRNA molecule, carrying the methionine amino acid that will serve as the first amino acid of the polypeptide chain, is bound to the P site on the ribosome. The A site is aligned with the next codon, which will be bound by the anticodon of the next incoming tRNA. Next, peptide bonds between the now-adjacent first and second amino acids are formed through a peptidyl transferase activity.
For many years, it was thought that an enzyme catalyzed this step, but recent evidence indicates that the transferase activity is a catalytic function of rRNA Pierce, After the peptide bond is formed, the ribosome shifts, or translocates, again, thus causing the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to pick up another amino acid.
In addition, the A site is now empty and ready to receive the tRNA for the next codon.
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