Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3.
Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV-geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA - formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site - are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.
Yoo
Jae-Ho
JH
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
RajBhandary
Uttam L
UL
D017104:Bacteriophage M13 / Q000254:growth & development; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000235:genetics / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D029968:Escherichia coli Proteins; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D012358:RNA, Transfer, Met; D049409:Luciferases, Firefly; D015500:Chloramphenicol O-Acetyltransferase