Abstract
Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present.
Highlights
Translation Initiation Factor 2 (IF2; for a review, see [1]) is an essential cellular protein that brings mRNA, the 30S ribosome, and the initiator fMet-tRNA together into the 30S initiation complex and promotes association with the 50S ribosomal unit to form the 70S initiation complex [2,3,4]
The reaction in vitro requires the E. coli replication restart proteins PriA, PriC, and DnaT but not PriB, indicating that the mode of Mu replication reconstituted in this system is through the PriA-PriC restart system [18,19]. (The PriA-PriC pathway is one of the two major cellular restart pathways, the other being the PriA-PriB pathway, which requires PriA, PriB, and DnaT [18].) only truncated forms of IF2 (IF2-2 and IF2-3; Mr of 79.7 and 78.8 k compared to 97.3 k for full-length IF2-1), synthesized from two internal, in-frame start codons within the infB gene, have been found to be active in this in vitro system
Translation Initiation Factor 2 (IF2) is a bacterial protein that plays an essential role in the initiation of protein synthesis
Summary
Translation Initiation Factor 2 (IF2; for a review, see [1]) is an essential cellular protein that brings mRNA, the 30S ribosome, and the initiator fMet-tRNA together into the 30S initiation complex and promotes association with the 50S ribosomal unit to form the 70S initiation complex [2,3,4]. We have previously identified it as an essential component for reconstituting bacteriophage Mu replication by transposition in vitro, a process in which IF2 makes way for initiation of DNA synthesis by the cellular restart proteins [5]. This finding raises the question whether IF2 could play an important function in the maintenance of genome integrity by regulating the engagement or activity of restart proteins. The sulA mutation did not fully revert ,infB(del1). to the wild-type phenotype
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