We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA fMet to the aa-tRNAs Phe-tRNA Phe , AlatRNA Ala , Gly-tRNA Gly , Pro-tRNA Pro , Asn-tRNA Asn , and Ile-tRNA Ile , selected to cover a large range of intrinsic pK a -values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK obs a , at which the rate was half maximal. The pK obs avalues were downshifted relative to the intrinsic pK a -value of aa-tRNAs in bulk solution. Gly-tRNA Gly had the smallest downshift, while Ile-tRNA Ile and Ala-tRNA Ala had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK a -values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.ribosome | kinetics | rate limiting step | accommodation | molecular dynamics T he ribosome promotes protein elongation by transfer of the nascent peptide chain from P-site peptidyl-tRNA to A-site aminoacyl-tRNA (aa-tRNA) ( Fig. 1) and translocation of messenger RNA (mRNA) and tRNAs. Peptide bond formation is initiated by a nucleophilic attack of the α-amino group of the amino acid, ester linked to the A-site tRNA, on the ester carbonyl carbon of the peptide chain linked to the 3′-oxygen of the P-site tRNA (Fig. 2). Biochemical data (1-3), crystallographic data (4-6), and molecular dynamics simulations (7,8) have shown that the 2′OH group of A76 of the P-site tRNA greatly accelerates peptide bond formation by providing a shuttle of the proton from the attacking α-amino group to the leaving 3′O of the deacylated P-site tRNA. The rate of ribosomal peptidyl transfer is further accelerated by a network of H-bonds involving water molecules and conserved bases in the peptidyl transferase center (PTC) of the ribosome (4,8,9).Peptidyl transfer requires that the α-amino group of the amino acid on the A-site tRNA is in charge neutral rather than in protonated NH þ 3 form (Fig. 2) (10). At 25°C the pK a -values of the α-amino groups of amino acids in bulk water range from 8.8 (Asn) to 10.6 (Pro) units (11). The pK a -values of the aa-tRNAs, approximated by the pK a -values of the corresponding amino acid methyl and ethyl esters (12)(13)(14), are downshifted in relation to those of the amino acids by two units, with 20°C values ranging from 6.8 (Asn) to 8.6 (Pro) ( Table 1). The rate of ribosomal peptidyl transfer might therefore be expected to vary differently with pH in the physiological range 6-8 for different A-site bound aa-tRNAs. The sensitivity of peptide bond formation to pH-variation in the 6-8 range has so far only been observed for aa-tRNA analogues but not for native aa-tRNAs (15). It has been suggested that the lack of pH sensitivity for aa-tRNAs is that their accommodation in the A site is so slow that the expected p...

We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy tradeoff for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA His and, as also seen in vivo, Glu-tRNA Glu. We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.protein synthesis | genetic code | misreading | error hot spots | kinetics T he genetic code has 61 sense codons encoding the 20 canonical amino acids and three stop codons encoding termination of peptide elongation. The sense codons in the ORFs of mRNAs are translated on ribosomes by aminoacylated tRNAs (1). Rapid synthesis of the bacterial proteome requires that aminoacyl-tRNAs (aatRNAs) in ternary complex (T 3 ) with elongation factor Tu (EF-Tu) and GTP bind rapidly with large k cat /K m values to ribosomal aatRNA sites (A sites) programmed with cognate codons. High quality of the proteome requires that aa-tRNAs read noncognate codons with small k cat /K m values so the frequency of amino acid substitution (missense) errors is small (2). This means that high population genetic fitness requires sufficiently small missense error frequency for high proteome quality, yet not so small as to seriously reduce the speed of cognate codon reading by the universal rateaccuracy trade-off for all substrate-selective enzymatic reactions (2-4). The rate-accuracy trade-off prescribes the efficiency (k cat /K m ) of cognate product formation to decrease with increasing accuracy of substrate selection, often in a linear fashion (5). The rate-accuracy trade-off depends on (i) the maximal possible discrimination between right and wrong substrate of an enzymatic selection step (the d value) and (ii) the fraction, α d, of the d value that is implemented by the enzyme (2): As α d increases toward 1 the efficiency of cognate product formation decreases toward zero.The existence of maximal accuracy limits (d values) in amino acid discrimination by an amino acid-selecting protein was suggested years ago by Linus Pauling (6). He proposed that these d values would be very small for pairs of similar amino acids. For discrimination between valine and isoleucine he estimated a d value of 10, leading to the proposal of high intracellula...

Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA(Phe). The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA(Phe) derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA(Phe). We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.

Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.ribosome | error correction | fidelity | EF-Tu | ternary complex

Our ability to directly relate results from test tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell-time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe in perfect agreement with previous indirect estimates, and that once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.