Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli.

Abstract

When Escherchia coli cells are transferred from 300C to 4?C, transcription from specific promoters recognized by RNA polymerase containing a32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of a!32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-acZ gene fusion. Synthesis of a a!32-iB-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation-namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.Exposure of cells to heat or other stress induces the synthesis of heat shock proteins (1, 2). In Escherichia coli, a shift from 30°C to 42°C induces heat shock genes transiently by activating transcription from promoters specifically recognized by RNA polymerase containing o32 (3,4), encoded by the rpoH (htpR, hin) gene (5, 6). Enhanced transcription from heat shock promoters is caused by a transient increase in the cellular level of &32 (7,8) as a result of increased synthesis and stabilization (8). The increased synthesis of a£32 has been thought to occur primarily at the translational level, because (i) the synthesis rate of 32, but not of rpoH mRNA, increases markedly upon temperature up-shift (9), (it) heat-induced synthesis of a &32-,B-galactosidase fusion protein depends on the translational initiation region of rpoH and not on the promoters (ref. 8; see below), and (iii) the rpoH mRNA level also increases upon temperature up-shift, but this increase is preceded by the increase of a32 synthesis (8, 10).To further substantiate translational control of &32 and gain insight into early events of the heat shock response, we examined expression of deletion and base substitution derivatives of rpoH-lacZ gene fusion during heat shock. We show that expression of gene fusion is transiently activated even in the absence of RNA synthesis, strongly supporting translational control. Evidence indicates that rpoH mRNA translation is normally repressed during steady-state growth and is transiently derepressed upon shift to higher temperature. Furthermore, such a thermal regulation probably involves an interplay of positive and negative cis-acting rpoH mRNA regions that correspond to the translational initiation region and an internal coding region, re...

Deletion analysis of cspA of Escherichia coli: requirement of the AT‐rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction

“…Therefore, it appears that the DB sequence is essential for the cold shock induction of cold shock genes, in particular during acclimation phase, while the DB sequences in non-cold shock genes may not be fully functional, probably because of constrains in the mRNA structures. A DB sequence has also been identified in the rpoH mRNA 2 bases downstream from the initiation codon, which is essential for the heat shock induction of rpoH expression (Nagai et al, 1991). Therefore, the DB sequences may play critical roles in the translational efficiency of mRNAs under numerous stress conditions in which ribosomal functions become impaired.…”

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

“…In E. coli, all chromosomally encoded mRNAs contain untranslated leader regions preceding the initiation codon+ However, several phage and transposon-encoded mRNAs are leaderless when expressed in E. coli (Tedin et al+, 1999)+ Previous work had shown that both ribosomal protein S1 together with initiation factors IF2 and IF3 (Moll et al+, 1998;Tedin et al+, 1999;Grill et al+, 2000) were important for initiation complex formation on leaderless mRNAs and that IF3 in particular discriminated against the authentic AUG initiation codon in the leaderless cI mRNA (Tedin et al+, 1999)+ Using either wild-type (pdb wt) or mutant (pdb*) cI-lacZ fusions (Shean & Gottesman, 1992), we have asked if the IF3 mutants analyzed here affect expression of this leaderless mRNA+ The data presented in Table 3 show that both the R99L and R131P IF3 mutants enhanced leaderless cI expression modestly whereas expression of two leadered mRNAs encoding RpoH-lacZ (Nagai et al+, 1991) and LysU-lacZ (Ito et al+, 1993) fusion proteins was unaffected+ Consistent with these results, previous experiments by Tedin et al+ (1999) showed that the same infC135 mutant used here increased the expression of a tetR-lacZ fusion substantially (sevenfold)+ This suggests that IF3 recognizes the authentic initiation codon on leaderless mRNAs as a nonstandard start codon and, in the absence of any stabilization afforded by the SD-anti-SD interaction found in most leadered mRNAs, IF3 destabilizes initiation complex formation on leaderless mRNAs+ Consequently, the activity of IF3 is an important element in determining the expression of leaderless mRNAs+…”

“…The 2 infC (srjA) mutants used here were obtained from Tim Haggerty and Susan Lovett, Brandeis University (Haggerty & Lovett, 1993)+ The ability of these mutants to stimulate nonstandard initiation events was explored in a pilot experiment using a ⌬(lac-pro) derivative of the infC135 strain+ These experiments showed that the infC mutation increased initiation from internal GUG and UUG codons in lacZ (see Results)+ To facilitate their subsequent analysis, both infC mutations were moved into the ⌬(lac-pro) strain, MC41+ An aroD::Tn10 derivative of MC41 was first constructed and was then transduced to Aro ϩ with phage P1 prepared on each of the infC mutants+ These strains were designated MC171 (infC134 ) and MC172 (infC135 )+ The presence of the infC mutation was assessed by the ability of selected transductants to stimulate initiations from internal GUG and UUG sites in a lacZ construct and by DNA sequencing of an infCcontaining PCR fragment amplified from these strains+ In addition, infC135 strains had a distinct growth defect and both mutants formed wrinkled colonies on LB agar plates containing calcium and high concentrations (2%) of glucose+ The pACYC184-derived plasmids containing lacZ genes with altered initiation codons have been described previously (O'Connor et al+, 1997)+ Additional constructs were made by ligating pairs of complementary oligonucleotides (containing the desired initiation codon) with EcoRI and HindIII overhangs into HindIII/EcoRI-digested pSG25 (O'Connor & Dahlberg, 1993)+ All plasmid constructions were verified by plasmid sequencing+ Plasmids containing internal initiation codons were constructed earlier for analysis of frameshifting by mutant tRNAs (O'Connor et al+, 1989)+ The relevant sequences of all lacZ plasmids used in this work are shown in Table 4+ Leaderless lacZ-cI fusion plasmids (Shean & Gottesman, 1992) were obtained from Max Gottesman, and rpoH-lacZ (Nagai et al+, 1991) and lysU-lacZ (Ito et al+, 1993) fusion plasmids were obtained from T+ Yura and Y+ Nakamura, respectively+ Plasmids expressing initiator tRNAs were derived from pRSVCATam1+2+5 (Varshney & RajBhandary, 1990) and also contained either a wild-type metY gene encoding the initiator tRNA or the indicated mutant tRNAs+ b-Galactosidase assays and purification Cells to be assayed for b-galactosidase activity were grown in minimal E medium (Vogel & Bonner, 1956) containing glucose (0+2%) thiamine, proline, and casamino acids (0+2%) together with any necessary antibiotics+ b-Galactosidase was assayed as described previously (O'Connor & Dahlberg, 1993;O'Connor, 1998)+ For protein purification, cells were grown in rich medium and disrupted with a French press+ b-Galactosidase was purified from the postribosomal supernatants of these lysates by immunoaffinity chromatography (5 Prime r 3 Prime, Boulder, Colorado) and sequenced as described previously (O'Connor et al+, 1989)+…”

Identification of the heat-shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti The GenBank accession numbers for the sequences reported in this paper are AF128845 (rpoH1) and AF149031 (rpoH2).

“…In response to a sudden increase in temperature or other stresses, the levels of σ$# rise transiently because of increased synthesis and protein stabilization. The induction of synthesis is mainly mediated by relief of translational repression due to a secondary structure in the mRNA (Morita et al, 1999 ;Nagai et al, 1991 ;Yuzawa et al, 1993), though the level of rpoH transcription also increases slightly (Erickson et al, 1987 ;Tilly et al, 1986). Stabilization occurs with the release of σ$# from a DnaK\DnaJ\GrpE chaperone complex as DnaK binds denatured proteins generated under stress conditions (Gamer et al, 1996).…”