Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits.
Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD byin vitrostudies. However, thein vivorelevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts inEscherichia coliand by complementation analyses using various deletion constructs, we show that while the CTD is essential forE. colisurvival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays forin vivoinitiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.IMPORTANCEInitiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work inEscherichia coliis in demonstrating that while the C-terminal domain alone sustainsE. colifor its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Shreya Ahana
SA
Ayyub
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Divya
D
Dobriyal
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India varshney@mcbl.iisc.ernet.in.|Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Umesh
U
Varshney
D018387:Codon, Initiator / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D039665:Prokaryotic Initiation Factor-3 / Q000737:chemistry* / Q000378:metabolism*; D000072417:Protein Domains; D054657:Ribosome Subunits / Q000235:genetics / Q000378:metabolism*
D018387:Codon, Initiator; D029968:Escherichia coli Proteins; D039665:Prokaryotic Initiation Factor-3
3GC base pairs; initiation with AUA; initiation with AUU; initiator tRNA