Michael Yarus

Rates of aminoacyl-tRNA selection at 29 sense codons in vivo.

We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate. By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli. For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis. Therefore, the frameshift assay probably measures this process in vivo. Observed rates for aminoacyl-tRNA selection span a 25-fold range. Therefore, the time required to transit different codons in vivo probably differs substantially. Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons. This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons. However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection. Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons. Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly. Other factors are therefore more important than speed for some codons. Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes. Instead, there is a slight preference for slower aminoacyl-tRNA selection. The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex. Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing. However, no more general relationship is evident between codon/anticodon composition and rate of aminoacyl-tRNA selection. The frameshift method can be extended to all codons.

Recognition of tRNA by isoleucyl-tRNA synthetase: Effect of substrates on the dynamics of tRNA-enzyme interaction☆

An interaction between the isoleucine catalytic site and the tRNA recognition site of isoleucyl-tRNA synthetase (Escherichia coli) was detected using an assay for the binding of tRNA to enzyme. The effect of isoleucine is to increase the rate at which tRNA enters and leaves its binding site by sixfold without, therefore, a large effect on the equilibrium constant for tRNA binding. This effect requires neither transfer of isoleucine to tRNA nor activation of isoleucine as isoleucyl-AMP; the binding of isoleucine alone suffices. It appears from these data that release of aminoacyl-tRNA is the rate-limiting step in the acylation, or transfer, reaction. The apparent maximum rate constant for association of tRNA and enzyme is relatively large, 6 × 106 m−1 sec−1 (17 °C).

On the properties and utility of a membrane filter assay in the study of isoleucyl-tRNA synthetase

Abstract The effects of several variables on an assay which utilizes nitrocellulose filters to detect the binding of ligands to proteins are explored and the utility of this assay is illustrated for two cases: ( a ) the combination of two macromolecules, tRNA Ile and isoleucyl-tRNA synthetase (IRS), and ( b ) the formation of the isoleucyl adenylate adduct of IRS. The standard thermodynamic quantities for the tRNA binding reaction were determined to be ΔF = −11.4 kcal/mole, ΔH = −1.8 kcal/mole, and ΔS = 33 cal/mole-degree at 17°C. Using the filter method to measure IRS(ile-AMP), one can follow transfer of the ile moiety to tRNA Ile ; the kinetics suggest that the breakdown of IRS(ile-AMP) upon exposure of the complex to tRNA Ile is not a result of failure of a normal step in the aminoacyl transfer reaction sequence.

Amino Acids as RNA Ligands: A Direct-RNA-Template Theory for the Code's Origin

Abstract. Numerous RNA binding sites for specific amino acids are now known, coming predominantly from selection-amplification experiments. These sites are chemically discriminating despite being predominantly small, simple RNA structures: internal and bulge loops. Recent studies of sites for hydrophobic side chains suggest that there are other generalizable structural features which recur in hydrophobic RNA sites. Further, sites for hydrophobic side chains can contain codons for the bound amino acid, as has also long been known for the polar amino acid arginine. Such findings are comprehensively reviewed, and the implications for the origin of coded peptide synthesis are considered. An origins hypothesis which accommodates all the data, DRT (direct RNA templating), is formulated.

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2.

It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.

A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis.

A 29-nt RNA catalyst successively forms the aminoacyl ester phe-RNA, and then peptidyl-RNA (phe-phe-RNA), given phenylalanine adenylate (phe-AMP) as substrate. Catalysis of two related reactions at similar rates supports the argument that RNA catalysts would evolve as groups with similar mechanisms. In particular, successive aminoacyl- and peptidyl-RNA synthesis by one RNA suggests that uncoded but RNA-catalyzed peptide synthesis would evolve before the synthesis of coded peptides.

Multiple translational products from a five-nucleotide ribozyme

An indispensable step in protein biosynthesis is the 2′(3′) aminoacylation of tRNA by aminoacyl-tRNA synthetases. Here we show that a similar activity exists in a tiny, 5-nt-long RNA enzyme with a 3-nt active center. The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2′-ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2′ Phe-RNA product can be elaborated into multiple peptidyl-RNAs. Reactions do not require divalent cations, and have limited dependence on monovalent cations. Small size and minimal requirements for regiospecific translational activity strongly support the hypothesis that minuscule RNA enzymes participated in early forms of translation.

How mitochondria redefine the code.

Abstract. Annotated, complete DNA sequences are available for 213 mitochondrial genomes from 132 species. These provide an extensive sample of evolutionary adjustment of codon usage and meaning spanning the history of this organelle. Because most known coding changes are mitochondrial, such data bear on the general mechanism of codon reassignment. Coding changes have been attributed variously to loss of codons due to changes in directional mutation affecting the genome GC content (Osawa and Jukes 1988), to pressure to reduce the number of mitochondrial tRNAs to minimize the genome size (Anderson and Kurland 1991), and to the existence of transitional coding mechanisms in which translation is ambiguous (Schultz and Yarus 1994a). We find that a succession of such steps explains existing reassignments well. In particular, (1) Genomic variation in the prevalence of a codons third-position nucleotide predicts relative mitochondrial codon usage well, though GC content does not. This is because A and T, and G and C, are uncorrelated in mitochondrial genomes. (2) Codons predicted to reach zero usage (disappear) do so more often than expected by chance, and codons that do disappear are disproportionately likely to be reassigned. However, codons predicted to disappear are not significantly more likely to be reassigned. Therefore, low codon frequencies can be related to codon reassignment, but appear to be neither necessary nor sufficient for reassignment. (3) Changes in the genetic code are not more likely to accompany smaller numbers of tRNA genes and are not more frequent in smaller genomes. Thus, mitochondrial codons are not reassigned during demonstrable selection for decreased genome size. Instead, the data suggest that both codon disappearance and codon reassignment depend on at least one other event. This mitochondrial event (leading to reassignment) occurs more frequently when a codon has disappeared, and produces only a small subset of possible reassignments. We suggest that coding ambiguity, the extension of a tRNAs decoding capacity beyond its original set of codons, is the second event. Ambiguity can act alone but often acts in concert with codon disappearance, which promotes codon reassignment.

On malleability in the genetic code

To explain now-numerous cases of codon reassignment (departure from the “universal” code), we suggest a pathway in which the transformed codon is temporarily ambiguous. All the unusual tRNA activities required have been demonstrated. In addition, the repetitive use of certain reassignments, the phylogenetic distribution of reassignments, and the properties of present-day reassigned tRNAs are each consistent with evolution of the code via an ambiguous translational intermediate.

RNA-ligand chemistry: a testable source for the genetic code.

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the codes origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.