Protein synthesis in E. coli
is a complex system in which 45 elongator aminoacyltRNA (aa-tRNA) substrates (Sprinzl et al., 1998
) quickly and accurately decode the 61 different sense codons on the ribosome. Each aa-tRNA is chemically distinct, consisting of different combinations of sequences, lengths, post-transcriptional modifications, and esterified amino acids. For decoding to occur, these very different molecules must bind the ribosome at the same discrete site and undergo several intermediate steps before entering the ribosomal A site where peptide bond formation takes place. It is generally accepted that all elongator aa-tRNAs go through the same pathway and thus encounter the same succession of defined ribosomal environments. However, it is unclear whether the individual aa-tRNAs transit the ribosome with identical thermodynamic and kinetic properties. For example, an aa-tRNA with a GC rich anticodon may initially bind tighter to its codon than an aa-tRNA with an AU rich anticodon. Similarly, a tRNA esterified to a small non-polar amino acid may pass from the entry site into the A site more rapidly than a tRNA esterified to a bulky aromatic amino acid.
Available data sets do not agree as to whether individual aa-tRNAs act similarly or differently during decoding. Several in vivo
experiments suggest that different aa-tRNAs and even synonymous codons are decoded at different rates. In one such experiment, an insertion of three identical test codons was placed in the leader sequence of a gene regulated by the pyrE
attenuator, thereby coupling translation and transcription and revealing a six fold difference in transcriptional attenuation for 12 different codons (Bonekamp et al., 1989
). Another study used pulse chase experiments to measure expression of β-galactosidase which contained an insert of eight identical test codons late in the lacZ
gene and found a five fold difference in the rate of protein production for four different codons (Sørensen and Pedersen, 1991
). Finally, a study which measured how effectively 29 different codons could compete with a common frameshift site, which served as a kinetic standard, found a 60 fold range of incorporation efficiency (Curran and Yarus, 1989
). While all of these experiments suggest that different aa-tRNAs are incorporated into the ribosome differently, they all measure the decoding rate indirectly so the observed differences may not necessarily reflect intrinsic kinetic or thermodynamic differences in how individual aa-tRNAs interact with the ribosome. In contrast, a limited number of biochemical experiments using purified E. coli
ribosomes suggest that aa-tRNAs may function similarly to one another. Similar dissociation rates of eight different aa-tRNAs from the ribosomal A and P sites suggest that they bind to the sites in a thermodynamically equivalent manner (Fahlman et al., 2004
). However, the very slow non-enzymatic release of aa-tRNAs from ribosomes may not provide insight into how tRNAs behave in the very fast decoding pathway. The only experiments that directly and quantitatively compared the performance of aa-tRNAs in kinetically relevant steps in the elongation cycle used only a few aa-tRNAs that were tested in different laboratories under slightly different reaction conditions (Thomas et al., 1988
; Pape et al., 1998
; Cochella and Green, 2005
; Kothe and Rodnina, 2007
; Ling et al., 2007
). However, these few experiments do suggest that different aa-tRNAs may be selected into the ribosome similarly.
The goal of this work was to directly compare the decoding properties of a group of chemically diverse aa-tRNAs under identical conditions by testing the initial binding to the entry site as well as the rates of EF-Tu GTPase activation and aa-tRNA accommodation into the A site. This should establish whether or not the ribosome is sensitive to the chemical and structural differences among aa-tRNAs during decoding.