Regulation Of Heat Shock Genes Research Articles

Transcriptional activation of heat-shock genes in eukaryotes.

Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes. In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level. The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes. Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction. This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes. While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE. Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements. However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes. Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner. The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed. Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.

Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli.

The addition of isopropyl thio-beta-D-galactoside (IPTG) to Escherichia coli cells containing multiple copies of the heat shock regulatory gene htpR (rpoH) under the control of an IPTG-inducible promoter (P-tac) induced 15 of the 17 polypeptides of the heat shock (HTP) regulon. The time course and magnitude of the induction closely resembled that caused by a shift to 42 degrees C. Nevertheless the two means of inducing the heat shock regulon differed in outcome. Cultures grown at 28 degrees C and induced by incubation at 42 degrees C for 15 min gave significant protection against a challenge temperature of 50 degrees C, but no protection was afforded by a 15-min IPTG treatment at 28 degrees C. It could be shown that there was no interference by IPTG with the development of thermotolerance at 42 degrees C. Also, treatment of a wild strain of E. coli with various toxic agents revealed no correlation between the development of thermotolerance and the induction of any subset of the heat shock proteins. Thermotolerance appears to develop by processes other than the htpR-dependent induction of heat shock proteins.

Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene.

In Escherichia coli the product of the rpoH (htpR) gene, sigma 32, directs RNA polymerase to initiate transcription from heat shock promoters at all temperatures. Transcription of the heat shock genes is increased when cells are exposed to high temperatures because of increased transcription initiation by sigma 32-RNA polymerase. As a step toward understanding the regulation of the heat shock response we have examined the transcription of the rpoH gene. Using S1 mapping, promoter cloning, and in vitro transcription, we have identified the promoters and the terminator for the rpoH transcription unit. The rpoH transcripts are monocistronic and originate from at least three promoters. None of the promoters is recognized by sigma 32-RNA polymerase. Two are recognized by sigma 70-RNA polymerase and are active at both low and high growth temperatures. We do not know what form of RNA polymerase recognizes the third promoter. Transcripts from this promoter are abundant only at high temperature and are present after shift to the lethal temperature of 50 degrees C, even at times when there are no detectable transcripts from the other rpoH promoters. The amount of rpoH mRNA increases fivefold by 8 min after shift from 30 to 43.5 degrees C but rpoH mRNA synthesis increases by less than twofold, indicating that there is post-transcriptional control of the level of rpoH mRNA and presumably of sigma 32.

The Escherichia coli heat shock regulatory gene is immediately downstream of a cell division operon: the fam mutation is allelic with rpoH.

Several mutations which affect critical cell functions in Escherichia coli map at 76 min on the chromosome. The genes which map in this region are the cell division genes fts Y, E, X and S, the heat shock regulatory gene rpoH/htpR/hin, the lipoprotein biogenesis gene fam and another essential gene dnaM. We determined the relative positions of most of these genes and show that the rpoH gene lies immediately downstream of the last gene (ftsX) of a cell division operon and is transcribed in the same direction. We also show that the fam-715 mutation is allelic with rpoH and so the conditional lipoprotein deficiency of the fam mutation must be due to the pleiotropic nature of the heat shock response.

Heat shock regulatory gene rpoH mRNA level increases after heat shock in Escherichia coli

The Escherichia coli rpoH gene product sigma 32 is essential for the increase in heat shock gene transcription found after exposure of the bacteria to a sudden temperature increase. It is not known how the concentration of active sigma 32 is modulated. We showed that rpoH transcript levels increased after heat shock and that the magnitude of the increase in the level of mRNA was correlated with the magnitude of the temperature shift. The increase in the level of rpoH mRNA was still found in rpoH mutants so the mechanism of induction differed from that of the set of previously identified heat shock genes. The increased concentration of rpoH mRNA should result in a higher level of sigma 32, which is likely to be important for increasing heat shock gene transcription.

Heat shock response in Escherichia coli influences cell division.

Analysis of a mutant in fam, a pleiotropic gene affecting cell division in Escherichia coli, revealed that this gene is probably identical to the heat shock regulatory gene htpR. The fam-715 mutant and different htpR mutants were found to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce the normal cellular heat shock response. These defects were all corrected in fam and htpR mutants by complementation with plasmids carrying intact htpR+ or by recombination between these mutant alleles and a plasmid carrying only a portion of htpR. These results implicate the E. coli heat shock system in the regulation of cell division and raise the question of a similar role in other organisms.

Developmental effects of chemicals and the heat shock response in Drosophila cells

Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps). More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins. This phenomenon is one of the most basic regulatory mechanisms in living organisms. Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult. Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress. In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures. Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia. A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures. We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells. Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons. These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells. Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps. Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.

Transcriptional control of the S10 ribosomal protein operon of Escherichia coli after a shift to higher temperature.

In the 5 to 10 min immediately following a shift from 30 to 42 degrees C, the differential synthesis rates of ribosomal proteins encoded by the 11-gene S10 operon are transiently decreased. This effect results largely from a two- to threefold decrease in the differential rate of transcription of the operon. The inhibition of mRNA synthesis is apparently due to two types of control: (i) initiation of transcription at the S10 promoter is inhibited and (ii) readthrough at the attenuator in the S10 leader is decreased. Both of these effects on transcription are independent of the heat shock regulatory gene, htpR. Furthermore, the inhibition of transcription is observed in both relA+ and relA cells, suggesting that the temperature-induced repression does not require the relA-dependent accumulation of guanosine tetraphosphate (ppGpp). However, recovery from the heat shock was slower in relA+ strains than in relA strains. None of the other ribosomal protein operons that we analyzed showed such a strong decrease in transcription after the heat shock.

A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli.

The Bacillus subtilis rpoD gene has been sequenced and the primary structure of its product deduced. The molecular weight calculated for the sigma 43 is 42,828. The DNA and protein sequences of sigma 43 exhibit extensive homology to the Escherichia coli rpoD gene and its sigma 70 product, especially in the C-terminal two-thirds of the sequence. Other proteins exhibiting partial homology with sigma 43 include the E. coli nusA protein, the E. coli htpR (heat-shock regulatory gene) product sigma 32, and specific DNA-binding proteins. No amino acid homology was found between sigma 43 and B. subtilis phage SP01 sigma gp28, phage T7 RNA polymerase, or E. coli DNA primase. The gene exhibits a strong ribosomal binding site and a typical rho-independent transcription terminator sequence. A typical transcription terminator sequence was not observed upstream from the sigma 43 gene. The sigma 43 gene may be part of an operon, resembling the situation found in the E. coli sigma operon.

Production of abnormal proteins in E. coli stimulates transcription of ion and other heat shock genes

The product of the lon gene in Escherichia coli, protease La, plays an important role in the degradation of abnormal proteins. To determine whether the presence of abnormal proteins stimulates expression of this gene, we examined its transcription using a lon-lacZ operon fusion. After the cells synthesized large amounts of aberrant polypeptides (e.g. following incorporation of the arginine analog, canavanine, or production of incomplete proteins with puromycin, or induction of translational errors with streptomycin), these cells showed a two- to threefold increase in lon--lacZ expression. Furthermore, synthesis of a single cloned protein, e.g. human tissue plasminogen activator, caused a similar increase in lon transcription. This induction of lon by abnormal proteins requires the heat shock regulatory gene htpR and was not seen in htpR- cells. Under these various conditions, other heat shock proteins were also induced. Thus, the appearance of aberrant cell proteins may be a common signal under many adverse conditions for the induction of cell protease (or proteases) and other heat shock proteins.

Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature.

Nonsense mutations affecting the positive regulatory gene (htpR) of heat shock response have been obtained in a strain of Escherichia coli carrying no suppressor. The mutants can grow only at temperatures below 34 degrees C-35 degrees C. Heat, ethanol, and coumermycin induce major heat shock proteins in the wild-type but not in the htpR mutants. In contrast, the level of heat shock proteins synthesized at low temperature is unaffected. The htpR gene product is thus required for induction of heat shock proteins by heat or other stresses but not for their "basal-level" synthesis. Nucleotide sequence has been determined for the wild-type and the mutant alleles of htpR. The coding region appears to consist of 852 nucleotide pairs that correspond to 284 amino acids. Sequences commonly considered as signals for transcriptional initiation and termination were found flanking the coding region. Within this region, six amber, one opal, and two missense mutations were identified; the nonsense mutations are scattered along the gene, some being very close to the presumed amino terminus. These results indicate that the absence of htpR gene product is directly responsible for the failure to respond to heat shock or other stresses and for the inability to grow at high temperature. We propose that htpR represents a new class of genes that are essential for growth only at high temperatures (greater than 35 degrees C). Implications of the sequence homologies found among htpR, rpoD, and nusA proteins are discussed.

Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli.

Upon a shift to high temperature, Escherichia coli increase their rate of protein degradation and also the expression of a set of "heat shock" genes. Nonsense mutants of htpR (also called hin), suppressed by a temperature-sensitive suppressor, show lower expression of heat shock genes at 30 degrees C and fail to respond to a shift to 42 degrees C. These mutants were found to have a lower capacity to degrade abnormal or incomplete proteins than that of wild-type cells. This reduction in proteolysis equals or exceeds that in lon mutants, which encode a defective ATP-dependent protease, protease La, and is particularly large in htpR lon double mutants. The activity of protease La was higher in wild-type cells than in htpR mutants grown at 30 degrees C and increased upon shift to 42 degrees C only in the wild type. To determine whether htpR influences transcription of the lon gene, a lon-lacZ operon fusion was utilized. Introduction of the htpR mutation reduced transcription from the lon promoter at 30 degrees C and 37 degrees C. This defect was corrected by a plasmid (pFN97) carrying the wild-type htpR allele. Induction of the heat shock response with ethanol had little or no effect in htpR mutants but stimulated lon transcription 2-3 fold in wild-type cells and htpR cells carrying pFN97. Thus, lon appears to be a heat shock gene, and increased synthesis of protease La under stressful conditions may help to prevent the accumulation of damaged cellular protein.

Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor

We have sequenced a cloned segment of E. coli chromosomal DNA that includes the heat shock regulatory gene htpR. This segment contains an 852 nucleotide open reading frame bounded by transcriptional and translational signals. Both in vivo and in vitro the cloned segment produces a single protein that migrates in gels with the cellular protein (F33.4) implicated as the htpR product. Properties of a cloned fragment of the coding sequence truncated at the promoter-distal end are consistent with this assignment. The htpR gene product appears homologous to the sigma factor of RNA polymerase, and the two proteins are predicted to have similar secondary structure. In addition, two regions of the predicted htpR product resemble protein-DNA contact points conserved in known DNA-binding proteins.

Regulation of heat-shock genes: a DNA sequence upstream of Drosophila hsp70 genes is essential for their induction in monkey cells.

Heat-shock genes coding for heat-shock protein 70 (HSP70) in Drosophila melanogaster were subcloned into an SV40/plasmid recombinant capable of replication in permissive monkey COS cells. Following transfection of COS cells, no significant amount of Drosophila hsp70 RNA was detected at 37 degrees C. In contrast, a heat-shock at 43 degrees C or arsenite poisoning at 37 degrees C induced the massive production of Drosophila hsp70 RNA of correct size and faithful 5' ends. After heat-shock, the efficiency of hsp70 transcription in COS cells containing 2-4 X 10(4) gene copies was found to be 15-30% of that measured in Drosophila, on a per gene basis. By testing a series of 5' deletion mutants in this inducible transcription assay it was found that a sequence less than 70 bp long, directly upstream of the hsp70 gene, was essential for the heat or arsenite induction of transcription.