Regulation of the Synthesis of Ribosomes and Ribosomal Components

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

“…RF3 is not essential at 37 8C in Escherichia coli (Grentzmann et al+, 1994;Mikuni et al+, 1994)+ The observed similarity between RF3 and elongation factors led to the hypothesis that RF3 might be replaceable by EF-Tu or EF-G at 37 8C, inviting speculation on the function of RF3 (Laalami et al+, 1996;Nakamura et al+, 1996; Buckingham et al+, 1997)+ EF-G enables ribosome recycling by RRF in vitro in the presence of GTP (Hirashima & Kaji, 1972)+ We show here that RF3 GTPase activity can replace EF-G GTPase activity in in vitro ribosome recycling+ Release of the mRNA and tRNA after termination provides physical evidence supporting the involvement of RF3 in the dissociation of the translation complex after termination+ Eventually, a translocation step catalyzed by RF3 and GTP after termination could release RF1 or RF2 from the A-site and, at the same time, transport the terminal tRNA to the E-site in much the same way EF-G does during chain elongation+ Finally, we find an in vitro activity of RF3 for dissociation of mRNA and fmet-tRNA in the absence of RF1 or RF2 (Table 2)+ This activity shows an RF3 dissociation function in the absence of translation termination and is observed only at high factor concentration+ This observation is in concordance with the fact that an RF3 Ϫ mutant has previously been characterized as a suppressor of a pth (peptidyl-tRNA hydrolase) thermosensitive strain (Grentzmann, 1994)+ Peptidyl-tRNA hydrolase is responsible for hydrolyzing peptidyl-tRNA that has been prematurely dropped off the ribosome during translation (Menninger, 1979)+ At nonpermissive temperatures, pth temperature-sensitive mutants die, presumably due to accumulation of peptidyl-tRNA+ If RF3 plays a role in drop off, an RF3 mutant might suppress this thermosensitivity by lowering the amount of prematurely released peptidyl-tRNA+ Further genetic experiments on the involvement of RRF and RF3 in ribosomal drop off, as well as in vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirm that RF3 and RRF are able to stimulate peptidyl-tRNA release from ribosomes (Heurgué-Hamard et al+, 1998) Our data showing release of a short messenger RNA in the presence of RRF may appear to be contradictory to the recently published report of Pavlov et al+ (1997) suggesting no release of short synthetic mRNA by RRF+ On the other hand, a very recent report from Janosi et al+ (1998) has proven the concept of RRF functioning in mRNA release from the posttranslational complex in vivo+ The major difference between our experiments and those of Pavlov et al+ is that their mRNA contains a strong Shine and Dalgarno sequence only a few nucleotides away from the termination codon+ Reinitiation of translation after termination, due to a ShineDalgarno sequence near a stop codon, is a phenomenon that has been reported previously as coupled translation in prokaryotes (for review see Nomura et al+, 1984;Oppenheim & Yanofsky, 1980)+ To prove a role of RF3 in ribosome recycling, physical evidence for mRNA release from the ribosome is necessary+ Our somewhat simpler in vitro assay is independent of a ShineDalgarno sequence and allows observing mRNA release by RRF as one expects with most naturally occurring mRNAs+ Our res...…”

The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli

“…There are several proteins, e.g., r-proteins, aminoacyl-tRNA ligases, EF-Tu, EF-G, and glucose-6-phosphate dehydrogenase (gnd), which are synthesized in a manner coordinate to that of rRNA (7,17,33). However, in each of these cases, it has been shown that the regulatory mechanism achieving this coordinate regulation is different from that governing the regulation of rRNA.…”

“…Thus, the regulation of expression of the trmA gene appears similar to that of rRNA genes. Unlike rRNA genes, the trmA gene responds to gene dose as would be expected according to the ribosome feedback model (33,36). In order to analyze the regulatory features of the trmA operon, we have determined the nucleotide sequence of the trmA gene in E. coli and the nucleotide sequence covering the promoter region of trmA from Salmonella typhimurium.…”

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

“…Percentage of mutant rRNA in the total cellular rRNA and the rRNA fractions of PEM100 pSTL102, JM83 pSTL102, PEM100 pKK1400U/2058G, and JM83 pKK1400U/2058G when grown at 30 8C (light gray) and 42 8C (dark gray) as determined by primer extension analysis+ Values are given for 16S rRNA (A and C) and 23S rRNA (B and D) in the total cellular rRNA pool (Tot), polysome fraction (Pol), free-subunit fraction (Sub) and 70S monosome fraction (70S)+ major determinant of growth rate under optimal conditions is cellular protein-synthetic capacity (Nomura et al+, 1984;Lindahl & Zengel, 1986)+ The increases in doubling time observed in a wild-type EF-G background (i+e+, in PEM100 at 30 8C or in JM83 at either 30 8C or 42 8C) reflect a reduced translational efficiency due either to overloading of ribosomes per cell by plasmidencoded rrn operons (pSTL102) (Gourse et al+, 1982) or to a defect in the decoding region of 16S rRNA (C1400U mutation)+ However, despite the detrimental effect on growth rate, both plasmids effectively suppress the ts EF-G mutation at 42 8C (Table 2)+ The sites of both suppressor mutants in 16S rRNA are located in the decoding region of the 30S subunit (Dahlberg, 1989)+ The site of the EF-G mutation, domain IV, binds to the ribosome in close proximity to C1400 (Agrawal et al+, 1998;Wilson & Noller, 1998a), and UV crosslinking experiments have revealed that position 1400 associates with the anticodon loop of the aa-tRNA in the ternary complex with EF-Tu-GTP (Prince et al+, 1982), of which EF-G domain IV is a structural mimic (Nissen et al+, 1995)+ Position 1192 in 16S rRNA is located in helix 34, which has also been placed in the decoding region of the ribosome (Dontsova et al+, 1992;Moine & Dahlberg, 1994)+ The resistance to spectinomycin that is conferred by the C1192U substitution suggests that this nucleotide is involved in the translocation step of elongation, as spectinomycin is believed to inhibit EF-G-ribosome interactions (O'Connor et al+, 1995)+ Furthermore, the spectinomycin-resistance phenotype conferred by pKK1192U can be suppressed by a mutation in EF-G (Johanson & Hughes, 1994)+ Interestingly, mutants in 16S rRNA alone are not sufficient to suppress the ts EF-G mutant; there is a need for the 23S rRNA mutation A2058G, which resides in the peptidyltransferase loop of domain V (Douthwaite, 1992)+ Both of these functional centers on the ribosome are directly involved in the elongation cycle (Wilson & Noller, 1998b), but mutations at the two known EF-G binding sites in 23S rRNA, 1067 and 2661, did not give suppression and, in fact, were incompatible with the EF-G mutation at any temperature (Table 1)+ The preferential incorporation of mutant 16S and 23S rRNA into the polysomes of PEM100 pSTL102 and PEM100 pKK1400U/2058G at 42 8C (Fig+ 1) suggests the suppressor mutations are more functional than wildtype rRNAs at the restrictive temperature+ It is clear, however, that the polysome fractions are not composed exclusively of mutant rRNA, but do contain some wild-type rRNA+ Suppression of the ts EF-G defect must be achieved by ribosomes containing either a 16S or a 23S rRNA mutation+ Mutat...…”