Specialized transducing phage for the initiation factor 3 gene in Escherichia coli.

Abstract

A previously isolated thermosensitive mutant [Springer, M., Graffe, M. & Grunberg-Manago, M. (1977) Mol. Gen. Genet. 151,[17][18][19][20][21][22][23][24][25][26] exhibits two defects in vitro, one in the initiation factor IF3 and the other in the L-phenylalanine: tRNAPh' ligase (EC 6.1.1.20). Specialized X transducing phages that transduced this mutant to thermoresistance were selected. In vitro studies showed that the transductants had a normal MF3 activity. One of these transducing phages was shown to code for a protein synthesized under the control of Escherichia coli promoters, which has the same molecular weight as IF3. This protein crossreacts specifically with MF3 antisera and comigrates with pure 1F3 in a two-dimensional gel system. In Escherichia coli genes for the translation apparatus are located at a number of sites which are scattered along the chromosome (1-3). Transducing X bacteriophages have proved to be very useful for unraveling the genetic organization of these sites (4-6). Until recently similar studies on the initiation of translation have been lacking and as a result this process has only been studied biochemically (7). A previous work described a thermosensitive mutant exhibiting two defects in vitro: a thermolabile initiation factor IF3 and a modified L-phenylalanine:tRNAPhe ligase (phenylalanyl-tRNA synthetase, EC 6.1.1.20) (8); these defects were shown to be located at 38 min on the new E. coli map (9). Moreover, stable heterodiploids (mutant chromosome/wild-type episome) were shown to have a thermoresistant phenotype (8). Therefore, thermoresistant transductants could be selected for, using specialized transducing phages that generally form merodiploids when integrated into the E. coli chromosome.The present work describes the isolation of a X bacteriophage that, when integrated in the mutant chromosome, transduces the strain to thermoresistance in vvo and reverses both defects in vitro. This phage is shown to code for a 22,000 molecular weight protein that is under the control of E. coli promoters. This protein is also shown to be specifically recognized by antiserum against IF3, and to comigrate with pure IF3 in a twodimensional gel system. MATERIAL AND METHODSE. coli and bacteriophage X strains are listed in Table 1 (Fig. 1, 1 (Fig. 1, 3) ,4Ci/Amol) per ml was added. After 35-min aeration at 430, the label was isotopically diluted with an equal volume of nonradioactive leucine at 5 mg/ml. The cells were then centrifuged 20 min at 20,000 X g and resuspended in 500 ,ul of the sonication buffer (Tris-HCI, pH 7.5, 50 mM/Mg acetate, 10 mM/ NH4C1, 1.5 M). Two 100-W sonications were performed at 0°A bbreviations: IF, initiation factor; Mr, molecular weight. 3970The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo

“…As shown in Fig. 2 kDa; this band corresponds to IF3, as reported previously (21,24). The anti-IF3 serum also recognized a band in the M. xanthus extract with an apparent molecular mass in the 30-kDa range (Fig.…”

“…7). The anti-IF3 serum reacted with IF3 (lanes 4 to 7), which has an apparent molecular mass of 22 kDa, similar to the molecular mass of 20.5 kDa predicted from its amino acid sequence (21,24 (7). Tyr-75 (Tyr-77 in M. xanthus) supports the specificity of the initiator tRNA-initiation codon interaction (7).…”

Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth.

“…These results also establish the absolute requirement offrr for E. coli growth. (33) proved that IF-3 is necessary for growth. As to the EFs, insertional inactivation of the tufA, but not of tufB, (genes for EF-Tu) by bacteriophage Mu is lethal and is complemented by plasmid-born tufA (34).…”

Translation initiation factor IF2 of the myxobacterium Stigmatella aurantiaca: presence of a single species with an unusual N-terminal sequence

“…The genes for all three factors, namely, infA (IF1), infB (IF2), and infC (IF3), were first cloned in Escherichia coli (30,36,43). These three proteins play essential roles in several steps of translation initiation, from the selection of the initiator tRNA (15) to the control of the entry into the elongation cycle (26).…”