Functional Significance of an Evolutionarily Conserved Alanine (GCA) Resume Codon in tmRNA in Escherichia coli

Abstract

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.Occasionally, during the process of translation, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. If not recycled, accumulation of the stalled ribosomes is detrimental to the organism as it sequesters not only the ribosomes but also the tRNAs (as peptidyl-tRNAs) and brings protein synthesis to a halt. In Escherichia coli and other eubacteria, a well-known pathway that recycles the stalled ribosomes utilizes tmRNA (SsrA or 10Sa RNA) by a process called trans-translation (18,36). The tmRNA contains a tRNA like domain and an mRNA region encoding a short peptide (AN DENYALAA in E. coli) followed by a termination codon (Fig. 1A). The tmRNA is aminoacylated with alanine (in its tRNAlike domain) and recruited to the A-site of the ribosome stalled on a truncated mRNA. The polypeptide from the P-site tRNA is transferred to the alanine on the tRNA-like domain of tm-RNA to form (peptidyl)-alanyl-tmRNA. The ribosome then resumes translation (trans-translation) decoding the first codon (the "resume codon") and the remaining codons of the short open reading frame in the tmRNA. When the termination codon in the tmRNA reaches the ribosomal A-site, the C-terminally tagged polypeptide is released. Thus, the process of trans-translation in E. coli appends AANDENYALAA sequence to the incomplete polypeptide and marks it for degradation by cellular proteases. The C-terminal amino acids in the appended sequence are important for recognition by the cellular proteases; and while the tmRNA mutants that encode ANDENYALDD or ANDEHHHHHH rescue the stalled ribosomes, they do not direct the tagged proteins to degradation (9,11,17,25). Interestingly, the first amino acid (Ala) of the short peptide encoded by the resume codon GCN is evolutionarily conserved across the tmRNA sequences from different species. In addition, although the ssrA mutants harboring alternate resume codons have been...

Overcoming stalled translation in human mitochondria

“…Aborted nascent peptides are transferred from the P-site tRNA to the alanine on this upstream tRNA-like structure, resulting in an (peptidyl)-alanyl-tmRNA. The downstream element of the tmRNA then acts as an mRNA, where the first triplet, or resume codon, generally encodes an alanine ( Kapoor et al, 2011 ). Protein synthesis is resumed with the addition of an approximately 10 amino acid tag, before terminating in a conventional stop codon.…”

Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis

“…Because the tag template after the +2 codon can be replaced by many different sequences without any loss of activity [ 24 , 29 ], it seems that the efficient tagging activity of the chimeric tmRNA M1: M. tuberculosis SmpB complex on E. coli ribosomes results from replacing the GCA-AAC sequence of the resume and +2 codon with the GCC-GAU sequence. Because changing the GCA triplet for the GCC triplet in the resume codon has very little effect on E. coli tmRNA tagging activity, the first nucleotide of the +2 codon is likely to enhance the tagging activity of chimeric tmRNA E1 in vitro [ 30 ]. This is consistent with a conservation of adenosine residues in the first two positions of the +2 codon [ 31 ].…”

When transfer‐messenger RNA scars reveal its ancient origins

“…The MLD is always located between PK1 and PK2 and encodes a specific amino acid sequence varying in length from 10 to 35 amino acid residues (ANDENYALAA in E. coli ), which is recognized by proteases and, therefore, triggers the destruction of incomplete polypeptides. The first codon GCA (alanine) is highly conserved, as is the upstream region necessary for the perfect positioning of the MLD. Interestingly, while this tmRNA structure is highly conserved in bacterial phyla, an uncommon form of a “two‐piece” tmRNA that breaks the mRNA domain loop has been observed in alphaproteobacteria and cyanobacteria, and rarely in some betaproteobacteria.…”