Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Abstract

(A) Predicted secondary structure of mRNA at the vemP-secD2 VA intergenic region. The RNA sequence from the fifth last codon of vemP to the third codon of secD2 VA are shown with the secondary structure predicted by CentroidHomfold. The putative SD sequence and the start codon of secD2 VA are indicated by box and underline, respectively. The P site and the A site of the VemP-stalled ribosome (Fig. 4C) are shown schematically. To separate the VemP arrest point and the secondary structure-forming region, we inserted one or two copies of the 18 nucleotides encoding the last five amino acid residues of VemP followed by the termination codon, shown at the bottom. (B) Separation of the arrest point and the stem-loop-forming region impairs the regulation. E. coli cells carrying indicated vemP-secDF2 VA plasmids (with or without the insertion mutations shown in A) were induced with IPTG for 15 min at 37°C and treated with (+) or without (−) 3 mM NaN 3 for 5 min. Cells were then pulse-labeled for 1 min. Cell extracts of equivalent radioactivities were subjected to anti-VemP (upper gel) and anti-V. SecD2 (lower gel) immunoprecipitation. In the upper gel, "p" indicates the signal peptide unprocessed form of VemP, which included the polypeptide part of the elongation arrested VemP, whereas "m" indicates the signal peptide-processed mature form. Relative intensities of V.SecD2 are shown in the bottom graph, taking the value of lane 1 as unity (n = 3). Gray and black bars represent results obtained without and with NaN 3 treatment, respectively. (C) The insertion mutations do not affect translation arrest of VemP. E. coli cells carrying pBAD24-Δss-vemP-V.secDF2 with or without the insertion mutations were induced with 0.02% arabinose for 1 h at 37°C. The same amounts of proteins were separated by 10% wide-range gel electrophoresis, followed by immunodetection of VemP. (D) The secD2 VA -secF2 VA interval. The nucleotide sequence from the fourth last codon of secD2 VA to the fifth codon of secF2 VA is shown. The predicted SD sequence of secF2 VA is underlined.

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis

“…Furthermore, our biochemical studies revealed the existence of a unique regulatory mechanism for V.SecDF2 in Vibrio alginolyticus (6) Our biochemical studies using an in vitro translation system with E. coli ribosomes elucidated the arrested state of VemP (6). A toe print assay indicated that the elongation arrest specifically occurs at the position where the Gln-156 codon encounters the P-site in the translating ribosome.…”

“…Separately, we also reported that most Vibrio species possess two sets of SecDF paralogs, namely, V.SecDF1 and V.SecDF2, that utilize Na + and H + motive forces, respectively, to stimulate protein translocation (6), providing a physiological role for monovalent cation conductance in SecDF function.…”

“…Thus, the peptidyl transfer reaction to Phe-157-tRNA in the A-site is blocked. The elongation arrest of VemP also occurred at the same position when a ribosome fraction prepared from the native organism V. alginolyticus was used (6). A systematic stop codon scan and Ala/Ser scan analyses using both E. coli and V. alginolyticus ribosomes showed that both ribosomes exhibit the same amino acid specificity for the elongation arrest of VemP.…”

“…A systematic stop codon scan and Ala/Ser scan analyses using both E. coli and V. alginolyticus ribosomes showed that both ribosomes exhibit the same amino acid specificity for the elongation arrest of VemP. As many as 17 amino acid residues in the C-terminal conserved region contribute to the elongation arrest (6); however, the exact role of each amino acid residue of the conserved region in VemP elongation arrest remains unknown.…”

“…These mutations are located at a constriction site in the ribosome exit tunnel. The former mutation dramatically decreased the VemP arrest efficiency, whereas the latter mutation did not; thus, specific interactions exist between the VemP nascent chain and the interior of the exit tunnel of the ribosome, and the interaction mode of VemP is different from that of SecM (6). However, the mechanism via which the conserved segment inactivates the peptidyl transfer center (PTC) 2 of the ribosome remains unclear.…”