Genetically encoded but nonpolypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall.
The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.
Muto
Hiroki
H
Institute for Virus Research, Kyoto University, Japan.
Nakatogawa
Hitoshi
H
Ito
Koreaki
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D011691:Puromycin / Q000494:pharmacology; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; C497347:SecM protein, E coli; D014157:Transcription Factors; D011691:Puromycin