Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes

Xin Qi Wu, Puneeth Iyengar, Uttam L. RajBhandary

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG. Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene. Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs. These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA. In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.

Original languageEnglish (US)
Pages (from-to)4734-4739
Number of pages6
JournalEMBO Journal
Volume15
Issue number17
StatePublished - Sep 2 1996

Fingerprint

RNA, Transfer, Met
Chloramphenicol O-Acetyltransferase
Ribosomes
Escherichia coli
Genes
Initiator Codon
Messenger RNA
Anticodon
Terminator Codon
Transfer RNA
Area Under Curve
Proteins

Keywords

  • Initiation complex
  • Initiator tRNA
  • Ribosomes

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes. / Wu, Xin Qi; Iyengar, Puneeth; RajBhandary, Uttam L.

In: EMBO Journal, Vol. 15, No. 17, 02.09.1996, p. 4734-4739.

Research output: Contribution to journalArticle

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AB - For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG. Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene. Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs. These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA. In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.

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