Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome

Ichikawa, Shinichi; Ryoji, Masaru; Siegfried, Zahava; Kaji, Akira

doi:10.1128/jb.171.7.3689-3695.1989

Cited by 15 publications

(10 citation statements)

References 39 publications

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“…3). The genes map-rpsB-tsf-smbA-fir are ordered clockwise; map codes for methionine amino peptidase (4), rpsB codes for protein S2 of the 30S ribosome subunit (2), tsf codes for protein chain elongation factor EF-Ts (2), and frr codes for ribosome-releasing factor (22).…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli

et al. 1992

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The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli. To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C. Ten of the mutants also showed cold-sensitive growth at 22 degrees C. Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned. The cloned suppressor gene complemented all of the 56 suppressor mutations. DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da. The gene product was indeed detected. The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E. coli chromosome, between the tsf and frr genes. The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C. Cells which lacked the SmbA protein ceased macromolecular synthesis. The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites.

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Section: Resultsmentioning

confidence: 99%

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli

et al. 1992

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“…Culture conditions and methods for cloning, ligation, and transformation were as described elsewhere (27,28) …”

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Identification of the promoter region of the ribosome-releasing factor cistron (frr)

Shimizu

Kaji

1991

J Bacteriol

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Previous studies of the structure and expression of the ribosome-releasing factor (RRF) cistron (frr) have suggested that an efficient promoter region is located in the RRF cistron. We report here on the nucleotide sequence and in vivo function of the RRF promoter. The transcriptional start site was determined by primer extension to be 58 bp upstream of the translational initiation codon offrr. The location of the RRF promoter region was confirmed by means of (i) deletion analysis of the 5' proximal sequences of frr fused to the chloramphenicol acetyltransferase reporter gene, (ii) analysis of RRF produced in vivo from the deletion derivatives offrr cloned into pUC19, and (iii) gel retardation analysis with Escherichia coli RNA polymerase. The -35 and -10 regions were TTacCc and TATAcT, respectively. The strength of the RRF promoter was similar to that of the lac promoter, as determined by in vivo expression of chloramphenicol acetyltransferase activity. However, the RRF promoter was not affected by the intracellular cyclic AMP level despite the presence of a cyclic AMP receptor protein binding site downstream of the RRF promoter.Ribosome-releasing factor (RRF) is an Escherichia coli protein responsible for the release of ribosomes from mRNA during the termination of protein synthesis (25). It has been demonstrated that the translational initiation site of the RRF gene (frr) is located at 4 min on the E. coli gene map, 1.1 kb downstream of the translational termination site of tsf (28). The DNA sequence offrr has been determined, and it was deduced that RRF consists of 185 amino acids with a calculated molecular weight of 20,639 (27). In addition, when frr was cloned into the vector pUC19 (45), E. coli harboring the resulting plasmid, designated pRR1, expressed RRF efficiently (27). This observation suggested that a site in the cloned fragment of pRR1 can serve as an efficient promoter for the high expression of RRF in combination with the high-copy-number vector (5). However, no information has been reported concerning the promoter for RRF expression, even though Bendiak and Friesen (4) proposed a possible location of a putative promoter for RRF expression downstream of tsf at 4 min on the E. coli gene map.In this communication, we report on the precise location and in vivo function of the RRF promoter. To make this determination, we constructed plasmids containing nested deletions in the upstream region of the RRF cistron by digestion with restriction enzymes and nuclease Bal 31. The deleted promoter regions were fused to a reporter gene encoding chloramphenicol acetyltransferase (CAT) and examined in vivo for expression of CAT activity. The RRF promoter region has been defined by sequencing each of the deletion-containing fragments and by gel retardation analysis of an E. coli RNA polymerase-DNA complex. The transcriptional start site has been determined by primer extension to be 58 bp upstream of the translational initiation codon offrr. The sequences of the -35 and -10 regions of the RRF promoter thus deter...

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“…RRF, discovered in 1970 (7) and confirmed independently in 1973 (40), is encoded by the frr gene, which was mapped to near 4 min, close to the gene encoding EF-Ts in Escherichia coli (14). The gene encoding RRF is found among all living organisms so far examined except for Archaea (see reviews listed above).…”

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“…Two factors, EF-G and ribosome recycling factor (RRF; formerly called ribosome releasing factor [19]), catalyze this step (19). In the absence of RRF, ribosomes not only remain on the mRNA but also resume unscheduled translation downstream of the termination codon (17, 33).RRF, discovered in 1970 (7) and confirmed independently in 1973 (40), is encoded by the frr gene, which was mapped to near 4 min, close to the gene encoding EF-Ts in Escherichia coli (14). The gene encoding RRF is found among all living organisms so far examined except for Archaea (see reviews listed above).…”

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Inhibitory Effect of Heterologous Ribosome Recycling Factor on Growth of Escherichia coli

Atarashi

Kaji

2000

J Bacteriol

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Ribosome recycling factor (RRF) of Thermotoga maritima was expressed in Escherichia coli from the cloned T. maritima RRF gene and purified. Expression of T. maritima RRF inhibited growth of the E. coli host in a dose-dependent manner, an effect counteracted by the overexpression of E. coli RRF. T. maritima RRF also inhibited the E. coli RRF reaction in vitro. Genes encoding RRFs from Streptococcus pneumoniae and Helicobacter pylori have been cloned, and they also impair growth of E. coli, although the inhibitory effect of these RRFs was less pronounced than that of T. maritima RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in T. maritima RRF differed totally from that of E. coli RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible for T. maritima RRF inhibition of the E. coli RRF reaction.Protein synthesis consists of three steps: initiation, peptide chain elongation, and termination with the release of the completed peptide chain. The step that follows these three steps, disassembly of the posttermination complex, is less well known but is essential for the next round of protein synthesis (for reviews, see references 15, 18, 20, and 21). Two factors, EF-G and ribosome recycling factor (RRF; formerly called ribosome releasing factor [19]), catalyze this step (19). In the absence of RRF, ribosomes not only remain on the mRNA but also resume unscheduled translation downstream of the termination codon (17, 33).RRF, discovered in 1970 (7) and confirmed independently in 1973 (40), is encoded by the frr gene, which was mapped to near 4 min, close to the gene encoding EF-Ts in Escherichia coli (14). The gene encoding RRF is found among all living organisms so far examined except for Archaea (see reviews listed above). Approximately 30 frr genes have so far been sequenced, partly in connection with bacterial genome sequencing projects (for example, see reference 2), and some RRFs have been characterized (22,29,32,44). In vitro protein synthesis is stimulated four-to eightfold by the addition of RRF (27,31,34). The GTP requirement (8,23), the fate of ribosomes at the termination complex in the presence of RRF (8, 23), and the possible role of IF3 in certain situations (4, 23, 28) have been studied. In addition to its role in ribosome recycling, RRF maintains translational fidelity (18). Although the exact mechanism of RRF action remains elusive, RRF has been proposed to bind to the ribosomal A site (20) because it reduces translational error and because its action is inhibited by the antibiotics which interfere with the A site (10). RRF competes for ribosomal binding with peptide release factors that are assumed to bind to the A site (5). RRF has a nearly perfect structural similarity to tRNA (36). We therefore proposed that RRF is translocated from the A site to the...

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Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome

Cited by 15 publications

References 39 publications

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli

Identification of the promoter region of the ribosome-releasing factor cistron (frr)

Inhibitory Effect of Heterologous Ribosome Recycling Factor on Growth of Escherichia coli

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