Release of Nonstop Ribosomes Is Essential

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-based trans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity. trans-translation is essential in some species, but in others, such as Caulobacter crescentus, trans-translation can be inactivated. To determine why trans-translation is dispensable in C. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lacking ssrA, the gene encoding tmRNA. One of these genes, CC1214, was essential in ΔssrA cells. Purified CC1214 protein could release nonstop ribosomes in vitro. CC1214 is a homolog of the Escherichia coli ArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in which ssrA has been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.

Protocol publication

[…] The protein sequence of CC1214 was used in a tblastn search () of the June 2014 NCBI database of representative microbial genomes. An E value cutoff of 10−7 and a molecular-mass cutoff of 20 kDa were used to identify homologs. In cases where a homolog was identified for a given phylum, that homolog was then used in a tblastn search of the remaining representative genomes in that phylum. If a homolog was not found, tblastn searches were performed with homologs from closely related phyla. A tree of Alphaproteobacteria species was constructed by collecting 16S rRNA gene sequences from PATRIC () and aligning them with BLAST. The tree was viewed and colored using Fig tree software (version 1.4.2 [http://tree.bio.ed.ac.uk/software/figtree/]). […]