Search for translation arrest peptides encoded upstream of genes for components of protein localization pathways

Abstract

Regulatory nascent peptides participate in the regulation of cellular functions by the mechanisms involving regulated translation arrest. A class of them in bacteria, called monitoring substrates, feedback-regulates the expression of a specific component of protein localization machinery. Three monitoring substrates, SecM, MifM and VemP have previously been identified. Here, we attempt at identifying additional arrest peptides in bacteria. Our bioinformatic searches over more than 400 bacterial genomic sequences for proteins that have the common characteristic features shared by the known monitoring substrates and subsequent in vitro and in vivo characterization of the highlighted sequences allowed the identification of three arrest peptides termed ApcA, ApdA and ApdP. ApcA and ApdA homologs are conserved among a subset of actinobacteria, whereas ApdP has homologs in a subset of α-proteobacteria. We demonstrate that these arrest peptides, in their ribosome-tethered nascent states, inhibit peptidyl transfer. The elongation arrest occurs at a specific codon near the 3′ end of the coding region, in a manner depending on the amino acid sequence of the nascent chain. Interestingly, the arrest sequences of ApcA, ApdA and ApdP share a sequence R-A-P-G/P that is essential for the elongation arrest.

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery

“…However, the stalling efficiencies on E. coli and B. subtilis ribosomes varied among each arrest peptide (Fig. 2 and 4), suggesting that the residues located upstream of the RAPP motif play an important role in determining the species specificity, as suggested previously for ApdA, and ApdP 28 . In addition, during the identification of YwcI, we found that most of the E. coli and B. subtilis proteins that contained RAPP-like motifs did not stall the ribosome (Supplementary Fig.…”

“…3c-f and Supplementary Fig. [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. For the arrest peptides that efficiently stalled both the E. coli and B. subtilis ribosomes, we also used the E. coli ribosome and confirmed that the estimated stalling site was identical.…”

“…We used the following search criteria, which we employed previously to identify ORFs encoding arrest peptides (Fig. 1b) 28 : (i) a uORF of the sec or yidC gene encoding a small protein with no annotated function; (ii) a uORF encoding an N-terminal secretion signal or a transmembrane (TM) sequence; (iii) a uORF encoding a C-terminal sequence conserved among its homologs; and (iv) a uORF encoding a spacer region between the localization signal and the C-terminal end of the conserved sequence with a size greater than 30 amino acid residues to ensure the exposure of the N-terminal localization signal outside of the ribosome when arrested. We extracted short ORFs of unknown function located upstream of the secA , secDF , and yidC genes and classified them into groups via clustering based on their amino acid sequences using the MMseqs2 function 30 .…”

uORF4u: a tool for annotation of conserved upstream open reading frames

“…This involves retrieval of the 5' upstream regions of protein coding genes of interest from sequence databases, and manual inspection of the region, with or without the aid of a sequence alignment to indicate functional conservation. Tell-tale signatures of potential uORFs are the presence of Shine-Dalgarno elements (in the case of prokaryotes), along with start and stop codons in the right context and frame (12)(13)(14). However, sequence gazing is a time-consuming and tedious task.…”

Geometric differences in the ribosome exit tunnel impact the escape of small nascent proteins

“…Various studies have hence focused on examining the impact of the biophysical properties of the exit tunnel on translation, highlighting key regions where the nascent chain interacts with the tunnel wall [11][12][13]. Among these key regions, critical interactions are susceptible to take place at the PTC [1,13], but also downstream at the so-called "constriction site" [12][13][14] located in the vicinity of the universally conserved ribosomal proteins uL4 and uL22 [15]. A further increase in the tunnel radius at the exit also defines a "vestibular" region, with enough space to accommodate tertiary structure [16,17].…”