Response to temperature shifts of expression of the amp gene on pBR322 in Escherichia coli K-12

Kuriki, Yoshitaka

doi:10.1128/jb.169.5.2294-2297.1987

Cited by 19 publications

(24 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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).…”

mentioning

confidence: 95%

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

1992

View full text Add to dashboard Cite

The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non-heat shock proteins. While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non-heat shock proteins is poorly understood. We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response. Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover. Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins.The heat shock response, broadly defined, describes the induction of a set of heat shock proteins and the corresponding suppression of the synthesis of non-heat shock proteins when cells are shifted from a low to a high temperature (18,20). In Escherichia coli, there are at least 20 heat shock proteins that are significantly induced upon a temperature shift (12). While the induction of heat shock proteins is mediated at the level of transcription by the cr32 factor encoded by the rpoH (htpR) gene (12,20), little is known about the mechanism(s) of suppression of non-heat shock proteins during the heat shock response (20). Zengel and Lindahl showed that the transient decrease in synthesis from the S10 ribosomal protein operon during a temperature shift from 30 to 42°C was regulated at the level of transcription initiation and by the extent of transcription beyond the S10 attenuator (26). 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). Heat shock apparently increased the rate of ribosomal protein mRNA degradation three-to fourfold, resulting in the decrease in the steady state levels of these mRNAs (14). Additional evidence suggests that heat shock induces a factor that is responsible for the increased degradation of ribosomal protein mRNAs (14).While there is no direct evidence that the aforementioned factor is an RNase, such a "heat shock RNase" could affect non-heat shock protein synthesis by selectively degrading non-heat shock protein mRNAs during the heat shock response. Since the functions of many of the known he...

show abstract

mentioning

confidence: 95%

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

1992

View full text Add to dashboard Cite

show abstract

“…Another possibility is that the amp promoter is occluded by the ompF promoter and thus has no influence on the level of P-galactosidase encoded by pNO5 and pNO18. When 3-galactosidase synthesis was initiated from the amp start codon, i.e., that taking place in cells harboring pNO5 or pNO18, the P-galactosidase level in cells growing at 42°C was only about 25% of that in cells growing at 30°C, as in the case of P-lactamase synthesis in cells carrying pBR322 (4). In contrast, cells harboring pNO2 produced about 1.2-fold more j-galactosidase at 42 than at 300C.…”

Section: Methodsmentioning

confidence: 92%

“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Kuriki

1989

J Bacteriol

Self Cite

View full text Add to dashboard Cite

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

show abstract

“…It was concluded that the temperature shift-up repressed P-lactamase synthesis at the translational level (4), in contrast to the characteristics of heat shock proteins, the syntheses of which are regulated by sigma 32 as the rpoH gene product at the transcriptional step (2,6,8,(12)(13)(14)(15). The repression of 1-lactamase synthesis by heat shock seems to be an example of the heat shock response of E. coli and should be useful for the study of the regulation of gene expression at the translational level in E. coli.…”

mentioning

confidence: 99%

Requirement of a heat-labile factor(s) for in vitro expression of the amp gene of pBR322

Kuriki

1987

J Bacteriol

Self Cite

View full text Add to dashboard Cite

The amp gene of pBR328 and pBR322 was expressed less efficiently at 45 than at 30°C in an in vitro coupled transcription-translation system of Escherichia coli. Preincubation of S30 extract at 45°C reduced specifically its ability to express the amp gene at 30C, indicating inactivation of a factor(s) required for efficient expression of the amp gene.I have recently reported that a shift-up of temperature from 30 to 42°C caused reversible repression of synthesis of pBR322-encoded P-lactamase in Escherichia coli but did not affect the level of 1-lactamase mRNAs. It was concluded that the temperature shift-up repressed P-lactamase synthesis at the translational level (4), in contrast to the characteristics of heat shock proteins, the syntheses of which are regulated by sigma 32 as the rpoH gene product at the transcriptional step (2,6,8,(12)(13)(14)(15) in precipitates with hot 5% trichloroacetic acid, was 3.8-fold faster at 45 than at 30°C. A portion of the radioactive products was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5) and subsequent fluorography (7). The products synthesized at 30°C gave two major bands that corresponded to the molecular weights expected for chloramphenicol acetyltransferase and pre-p-lactamase, respectively. On the fluorograph of the products synthesized at 45°C, however, chloramphenicol acetyltransferase was detected only as one major band (Fig. 1A). Similarly, when pBR322 (1) was used as a template instead of pBR3288 (11), the products at 45°C gave a much weaker band of pre-,-lactamase than those at 30°C (data not shown). Therefore, expression of the amp gene on the both plasmids was concluded to be efficient in the in vitro coupled transcription-translation system at 30°C but to be repressed at 45°C. For examination of whether the inefficiency of amp gene expression at 45°C was reversible, a reaction mixture without [35S]methionine and template DNA was incubated at 45°C for 6 min and then was shifted to 30°C, and protein synthesis was examined by adding [35S]methionine and pBR328 as a template. After this preincubation, the incorporation of [35S]methionine into the acid precipitate was reduced by about 30%. Analysis of the radioactive products by SDS-PAGE (5) and fluorography (7) showed that the incubation at 45°C before protein synthesis decreased markedly the ability to synthesize pre-,-lactamase at 30°C, but that this treatment hardly affected the synthesis of chloramphenicol acetyltransferase in the same reaction mixture (Fig. 1B). This effect of the preincubation at 45°C was further examined in more detail, and it was found that the activity to synthesize pre-p-lactamase decreased with a half-life of 3.5 min that was two-to threefold shorter than that for total protein synthesis in the in vitro system (data not shown). The result suggests that the in vitro expression of the amp gene requires some factor(s) that is more heat labile than the factors and enzymes involved in gene expression in general.To study the distribution of this putative...

show abstract

Response to temperature shifts of expression of the amp gene on pBR322 in Escherichia coli K-12

Cited by 19 publications

References 25 publications

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

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

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

Requirement of a heat-labile factor(s) for in vitro expression of the amp gene of pBR322

Contact Info

Product

Resources

About