The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli

Abstract

pBR322 contains the amp gene encoding ,-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, ,B-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of I"-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of Il-lactamase, the synthesis of I-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).Previous studies have shown that, in Escherichia coli harboring pBR322 or related plasmids that contain the amp gene, the synthesis of ,-lactamase encoded by this gene is transiently repressed by a temperature shift-up, e.g., from 30 to 42°C (4; unpublished results). This arrest of synthesis is followed by an adjustment of the cellular level of 3-lactamase to a new steady state (4). Thus, in E. coli as in eucaryotic cells (12,14), heat shock leads not only to transient overproduction of a specific set of (heat shock) proteins (10, 11), but also to transient repression of the synthesis of certain proteins (4, 7). Since the synthesis of 3-lactamase mRNA is not repressed by temperature shift-up (4), it is thought that the repression of ,B-lactamase synthesis occurs at the translation level. In fact, the translation of 1-lactamase mRNA in vitro has been shown to be specifically repressed at higher temperature, such as 45°C (unpublished results).The purpose of the present study is to identify the region of P-lactamase mRNA that is responsible for this repression. [araD139 A(ara-leu)7697 A(lac)X74 galU galK rpsL (Str+) ompRi01] were used as hosts for plasmids pAT153 (16) and pORFI (17), respectively. These plasmids were prepared by the method described before (8) and purified by equilibrium centrifugation in a CsCl2 gradient (8).Medium. L-broth (8) was used. When necessary, ampicillin and X-Gal dissolved in N,N-dimethylformamide (10 mg/ml) were added to L-broth agar at concentrations of 100 and 20 pLg/ml, respectively. Construction of recombinant plasmids. All manipulations of DNA were carried out by standard methods (8). The procedures used for construction of pNO5 and pNO2 are outlined in Fig. 1

Role of the heat shock response in stability of mRNA in Escherichia coli K-12

“…However, they also concluded that this regulation was independent of the rpoH allele (26). Kuriki showed that the heat shock-induced suppression of ,-lactamase synthesis was regulated by the initiation of translation and depended on a short nucleotide sequence containing the Shine-Dalgarno sequence and the ATG start codon of ,-lactamase (16,17).In contrast, there is evidence from yeast cells that mRNA degradation can affect the suppression of the synthesis of non-heat shock proteins during the heat shock response. A temperature shift from 23 to 36°C resulted in a decrease in the synthesis of ribosomal proteins, with a corresponding rapid decrease in the cellular levels of ribosomal protein mRNAs (14).…”

“…However, they also concluded that this regulation was independent of the rpoH allele (26). Kuriki showed that the heat shock-induced suppression of ,-lactamase synthesis was regulated by the initiation of translation and depended on a short nucleotide sequence containing the Shine-Dalgarno sequence and the ATG start codon of ,-lactamase (16,17).…”

Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1

“…The most preferred sequence of strong RBS in E . coli is believed to be [ 23 ]. To enhance the expression of the α-subunit and NhlE, the putative RBS ( ) between the nhlB and nhlA genes ( Fig 1A ) was replaced with an enhanced RBS ( ), and the spacing sequences between the two genes were shortened from 63 bp to 14 bp, constructing plasmid pET24a- nhlBrbsAE .…”

Different promoters of SHV-2 and SHV-2a -lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers

“…Both promoters and the intervening regions downstream of the start codons show no significant similarity to those of the TEM /?-lactamases (Russell & Bennett,198 l), to inducible promoters of chromosomally encoded /?-lactamases (Lindberg & Normark, 1986;Forsman et af., 1989) or to regulatory sequences sensitive to heatIabile protein, which may couple the /?-lactamase expression to the SOS response (Kuriki, 1989).…”