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Quantitative views of cellular functions require precise measures of rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here, we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli data set transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. Members of multiprotein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles for optimizing design. These include how the level of different types of transcription factors is optimized for rapid response and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. Our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from quantitative analyses of protein synthesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Molecular biology studies the cause-and-effect relationships among microscopic processes initiated by individual molecules within a cell and observes their macroscopic phenotypic effects on cells and organisms. These studies provide a wealth of information about the underlying networks and pathways responsible for the basic functionality and robustness of biological systems. At the same time, these studies create exciting opportunities for the development of quantitative and predictive models that connect the mechanism to its phenotype then examine various modular structures and the range of their dynamical behavior. The use of such models enables a deeper understanding of the design principles underlying biological organization and makes their reverse engineering and manipulation both possible and tractable The heat shock response presents an interesting mechanism where such an endeavor is possible. Using a model of heat shock, we extract the design motifs in the system and justify their existence in terms of various performance objectives. We also offer a modular decomposition that parallels that of traditional engineering control architectures.

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.

The Escherichia coli periplasmic peptidyl-prolyl isomerase (PPIase) SurA is involved in the maturation of outer membrane porins. SurA consists of a substantial N-terminal region, two iterative parvulin-like domains and a C-terminal tail. Here we show that a variant of SurA lacking both parvulin-like domains exhibits a PPIase-independent chaperone-like activity in vitro and almost completely complements the in vivo function of intact SurA. SurA interacts preferentially (>50-fold) with in vitro synthesized porins over other similarly sized proteins, leading us to suggest that the chaperone-like function of SurA preferentially facilitates maturation of outer membrane proteins.

The activity of the stress-responsive sigma factor, sigma(E), is induced by the extracytoplasmic accumulation of misfolded or unfolded protein. The inner membrane protein RseA is the central regulatory molecule in this signal transduction cascade and acts as a sigma(E)-specific anti-sigma factor. Here we show that sigma(E) activity is primarily determined by the ratio of RseA to sigma(E). RseA is rapidly degraded in response to extracytoplasmic stress, leading to an increase in the free pool of sigma(E) and initiation of the stress response. We present evidence that the putative inner membrane serine protease, DegS, is responsible for this regulated degradation of RseA.

The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation., (Copyright 1998 Academic Press)

SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.

The activity of the alternate sigma-factor sigmaE of Escherichia coli is induced by several stressors that lead to the extracytoplasmic accumulation of misfolded or unfolded protein. The sigmaE regulon contains several genes, including that encoding the periplasmic protease DegP, whose products are thought to be required for maintaining the integrity of the cell envelope because cells lacking sigmaE are sensitive to elevated temperature and hydrophobic agents. Selection of multicopy suppressors of the temperature-sensitive phenotype of cells lacking sigmaE revealed that overexpression of the lipoprotein NlpE restored high temperature growth to these cells. Overexpression of NlpE has been shown previously to induce DegP synthesis by activating the Cpx two-component signal transduction pathway, and suppression of the temperature-sensitive phenotype by NlpE was found to be dependent on the Cpx proteins. In addition, a constitutively active form of the CpxA sensor/kinase also fully suppressed the temperature-sensitive defect of cells lacking sigmaE. DegP was found to be necessary, but not sufficient, for suppression. Activation of the Cpx pathway has also been shown to alleviate the toxicity of several LamB mutant proteins. Together, these results reveal the existence of two partially overlapping regulatory systems involved in the response to extracytoplasmic stress in E. coli.

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, sigma(E). sigma(E) activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that sigma(E) activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25-fold induction of sigma(E) activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits sigma(E)-directed transcription in vitro, indicating that RseA acts as an anti-sigma factor. Deletion of rseB leads to a slight induction of sigma(E), indicating that RseB is also a negative regulator of sigma(E). RseB is a periplasmic protein and was found to co-purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on sigma(E) through RseA. Deletion of rseC, in contrast, has no effect on sigma(E) activity under steady-state conditions. Under induction conditions, strains lacking RseB and/or C show wild-type induction of sigma(E) activity, indicating either the presence of multiple pathways regulating sigma(E) activity, or the ability of RseA alone to both sense and transmit information to sigma(E).

The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways. Our previous finding that SecB-deficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway. Using depletion studies, we now demonstrate a requirement for GrpE in this pathway. In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and beta-lactamase), they are not the primary chaperones in this process.

Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves. As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues. We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E). With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit. Allele specific suppression is a genetic criterion for protein-protein interaction. Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation. Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide. We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme.

Control of RNA polymerase is a common means of regulating gene expression. A detailed picture of both the structure and how the structural details of RNA polymerase encode function is a key to understanding the molecular strategies used to regulate RNA polymerase. We review here data which ascribes functions to some regions of the primary sequence of the subunits (alpha, beta beta' sigma) which make up E. coli RNA polymerase. We review both genetic and biochemical data which place regions of the primary sequence that are distant from one another in close proximity in the tertiary structure. Finally we discuss the implications of these findings on the quaternary structure of RNA polymerase.

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

We previously reported that a region of the Escherichia coli chromosome at 18 min increased E sigma E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618-2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in E sigma E activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, E sigma E activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, E sigma E activity was dependent on expression of OmpX in certain strain backgrounds.

Streptolydigin (stl), a bacteriostatic inhibitor of transcription elongation, interacts with the beta subunit of Escherichia coli RNA polymerase. We have defined the target for stl resistance using chemical mutagenesis and mutagenic polymerase chain reaction. Mutations resulting in stl resistance are confined to a small cluster of contiguous amino acids, amino acids 543 to 546. These stlr mutants differ from one another in their levels of resistance to stl in vivo and in vitro. We have analyzed two of the mutants, A543V and F545S, for their effects on elongation and termination in vivo and in vitro. Neither affected termination at rho-dependent or rho-independent terminators. These mutants were indistinguishable from wild type in a T7 in vitro elongation assay. F545S, however, did exhibit slower elongation kinetics in a lambda tR1 pausing assay. We conclude that mutations in the stlr region can influence transcription elongation, but that these amino acids are not directly involved in catalysis.

sigma E and sigma 32 are two heat- and ethanol-inducible sigma-factors in Escherichia coli. The sigma 32 regulon is also induced by unfolded and misfolded proteins in the cytoplasm, and the function of many of the proteins in the sigma 32 regulon is to bind to cytoplasmic proteins and assist them in folding or unfolding. To further understand the function of the sigma E regulon, we searched for mutants that affected sigma E activity. Our results indicate that a signal generated by expression of outer membrane proteins modulates sigma E activity. Specifically, sigma E activity is induced by increased expression of OMPs and is reduced by decreased expression of OMPs. In addition, mutations that cause misfolded OMPs induce sigma E activity. This signal is generated after the fate of OMPs and periplasmic proteins diverge in the secretory pathway and is not the result of an accumulation of OMP precursors in the cytoplasm. Our results indicate that this effect of OMPs is specific to the sigma E regulon, because none of the above mutations affect sigma 32 activity. We propose that the sigma E regulon is involved in processes that occur in extracytoplasmic compartments and that these two heat-inducible regulons may have distinct but complementary roles of monitoring the state of proteins in the cytoplasm (sigma 32) and outer membrane (sigma E).

The accumulation of secretory protein precursors, caused either by mutations in secB or secA or by the overproduction of export-defective proteins, results in a two- to fivefold increase in the synthesis of heat shock proteins. In such strains, sigma 32, the alternative sigma factor responsible for transcription of the heat shock genes, is stabilized. The resultant increase in the level of sigma 32 leads to increased transcription of heat shock genes and increased synthesis of heat shock proteins. We have also found that although a secB null mutant does not grow on rich medium at a temperature range of 30 to 42 degrees C, it does grow at 44 degrees C. In addition, we found that a secB null mutant exhibits greater thermotolerance than the wild-type parental strain. Elevated levels of heat shock proteins, as well as some other non-heat shock proteins, may account for the partial heat resistance of a SecB-lacking strain.

Rifampicin-resistant (Rifr) mutations of Escherichia coli map to the central portion of the rpoB gene, which encodes the beta subunit of RNA polymerase. These mutations are located in three distinct clusters, designated I, II and III. Three intragenic suppressors of the cluster III Rifr mutation, rpoB3406(RH687), restore the ability of the mutant strain to grow at low and high temperatures and map to a single locus in cluster I. These suppressors are identical to two previously characterized Rifr alleles, rpoB3401(RC529) and rpoB3402(RS529). None of the other 14 previously identified Rifr mutations that we have characterized confers this phenotype. We suggest that this allele-specific suppression results from interaction between Cluster I and Cluster III of the beta subunit.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli. Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F). Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.

A set of 37 mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, was isolated using a selection for high constitutive expression of heat shock proteins. Of these, 11 mutants were able to carry out some but not all functions of DnaK. These partial function mutants were divided into two classes. Class I mutants are recessive and permit replication of bacteriophage lambda and growth of cells up to 40 degrees C. Class II mutants are dominant, do not permit growth of lambda, and are temperature-sensitive for growth above 34 degrees C. Mutations in both classes alter amino acids that are highly conserved in the 70-kDa heat shock protein family. The dominant negative mutations provide strong genetic evidence that at least one form of DnaK is multimeric. Moreover, every dominant negative mutation occurs at an amino acid that has been hypothesized to be intimately involved in the process of ATP binding and hydrolysis. Our findings provide strong support for the hypothesis that such mutations are excellent tools for identifying amino acids that play critical roles in protein function.

In Escherichia coli secreted proteins must be maintained in an export-competent state before translocation across the cytoplasmic membrane. This function is carried out by a group of proteins called chaperones. SecB is the major chaperone that interacts with precursor proteins before their secretion. We report results indicating that the DnaK and DnaJ heat shock proteins are also involved in the export of several proteins, most likely by acting as their chaperones. Translocation of alkaline phosphatase, a SecB-independent protein, was inhibited in dnaK- and dnaJ- mutant strains, suggesting that export of this protein probably involves DnaK and DnaJ. In addition, DnaK and DnaJ play a critical role in strains lacking SecB. They are required both for viability and for the residual processing of the SecB-dependent proteins LamB and maltose-binding protein (MBP) seen in secB null strains. Furthermore, overproduction of DnaK and DnaJ permits strains lacking SecB to grow in rich medium and accelerates the processing of LamB and MBP. These results suggest that under conditions where SecB becomes limiting, DnaK and DnaJ probably substitute for SecB and facilitate protein export. This provides the cell with a mechanism to overcome a temporary imbalance in the secretion process caused by an abrupt expansion in the pool of precursor proteins.

The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature. We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase. We show that ts8 and a second fda mutation, h8, isolated and characterized by A. Böck and F. C. Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.

The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown.

We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2. A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.

We have investigated the sequence requirements for the translational regulation of sigma 32 by examining the behavior of a new rpoH-lacZ protein fusion containing a short N-terminal fragment of sigma 32 fused to beta-galactosidase. Although the fusion retains rpoH translational initiation signals, it lacks translational regulation, implicating coding sequences within rpoH in this regulatory process.

Rifampicin-resistant (Rifr) mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have examined the effect of each of the 17 sequenced Rifr mutations in our collection on transcription termination. The effect of each Rifr mutation was measured at three types of terminators: simple terminators requiring only RNA polymerase to terminate in vitro, and complex terminators requiring either Rho or Tau for in-vitro termination. Almost every Rifr allele examined (14/17) affected readthrough at one or more of these terminators. We found that mutations with similar termination phenotypes were clustered suggesting functional specialization within the region of rpoB defined by the Rifr mutations. The interaction of the Rifr mutations with the defective rho15 allele was also investigated. Only two Rifr mutations suppress the termination defect of rho15 strains. We discuss models to explain how this region of the beta polypeptide might be involved in the process of transcription termination.

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.

A temperature-sensitive mutant sigma subunit (rpoD800) purified from Escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees C whereas wild type sigma remained stable up to 49 degrees C. Both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains. Conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensitive sigma from heat inactivation. Full activity was recovered from inactivated temperature-sensitive sigma aggregates by incubation in a buffer containing 6 M guanidine HCl and subsequent removal of denaturant by dilution. Both wild type and temperature-sensitive sigma recovered full activity levels, retaining their characteristic thermal inactivation temperatures after denaturation in 6 M guanidine HCl and renaturation. Transcription of T4 DNA by RNA polymerase containing the rpoD800 mutant sigma subunit remained undiminished for 10 min after shift up to 46 degrees C but was almost completely inhibited within the following 10 to 15 min.

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.

Recombinant DNA techniques allow rapid construction and characterization of tools for genetic dissection of essential genes. In this report, we describe construction of a set of deletions in rpoD, the gene which encodes the sigma subunit of E. coli RNA polymerase. We used this deletion set to identify a region of the sigma polypeptide which is dispensable for its function.

The rpoD gene encoding the sigma subunit of E. coli RNA polymerase is cotranscribed with rpsU and dnaG, encoding ribosomal protein S21 and DNA primase, respectively. After temperature upshift, a heat shock promoter (Phs) located within dnaG is transiently induced, causing increased transcription of rpoD. The extent of induction is sufficient to account for the heat shock response of sigma synthesis. The initiation site of this promoter was located about 360 bp upstream of rpoD by promoter cloning and S1 nuclease mapping. Plasmid deletions generated with Bal 31 nuclease show that the DNA sequence CTGCCACCC in the -44 to -36 region of this promoter is necessary for its heat shock activity. Heat induction of transcription from Phs is under the control of HtpR, a positive regulator of the heat shock response.

We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon tranfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45 degrees C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9 degrees C lower than does the wild type. The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E. coli RNA polymerase.

The htpR gene of E. coli encodes a positive regulator of the heat-shock response. We have fused the htpR gene to the inducible PL promoter of phage lambda. Overproduction of HtpR following a temperature upshift resulted in the overexpression of heat-shock proteins. We describe the purification and initial in vitro characterization of the factor controlling expression of heat-shock genes. The factor was the 32 kd htpR gene product. In vitro, a mixture of HtpR and core RNA polymerase initiated transcription at heat-shock promoters. The sigma factor encoded by rpoD was not required for this reaction. Therefore, HtpR is a sigma factor that promotes transcription initiation at heat-shock promoters. We propose that htpR be renamed rpoH and that the gene product be called sigma-32.

The sigma subunit of E. coli RNA polymerase is encoded by the rpoD gene. Within the sequence upstream from rpoD, we have identified the structural genes rpsU and dnaG, which encode the 30S ribosomal protein S21 and DNA primase, respectively. The three genes are in the order rpsU, dnaG rpoD, and are all encoded by the same DNA strand. Analysis of in vivo transcripts from this region shows that these genes are all within the same operon. By correlating the 5' and 3' ends of in vivo transcripts with our DNA sequence, we have identified several regulatory features of the operon. These features include tandem promoters upstream from rpsU, a terminator between rpsU and dnaG, an RNA processing site separating dnaG and rpoD, and the operon terminator just downstream from rpoD. Immediately upstream of the operon promoters is an active promoter for an unidentified gene. We discuss the regulatory significance of the operon features and the biological significance of an operon encoding proteins essential for translation, replication and transcription.

The Escherichia coli strain, ts-rnp5, originally described in 1975 by G. D. Burdick and H. Berger, is shown to possess an RNA polymerase (RNA nucleotidyltransferase) sigma subunit with an activity 4--6 times less thermostable at 45 degrees than sigma from wild-type strains. This defect remains associated with the sigma polypeptide through a variety of purification stages, including renaturation of sigma after its elution from sodium dodecyl sulfate/polyacrylamide gels. The mutation responsible for decreased thermostability of sigma, called rpoD1, cotransduces with dnaG and therefore is located at about 66 min of the E. coli genetic map.

The rpoD gene (encoding the 70,000 Mr sigma subunit of Escherichia coli RNA polymerase) is the most distal gene in an operon that contains three genes. The promoter-proximal gene is rpsU (encoding ribosomal protein S21) and the middle gene is dnaG (encoding DNA primase). During the stringent response, caused by a deficiency in an aminoacyl-tRNA, expression of rpsU is decreased, while expression of rpoD is not. This disco-ordinate regulation is due to increased transcription from a minor promoter upstream from rpoD, in the dnaG gene. Transcription from this promoter is also increased during the heat shock response. Expression of other heat shock proteins was found to increase during the stringent response. Thus, the stringent response in E. coli induces expression of heat shock proteins. The requirements for this stringent induction of the heat shock proteins differ from those for temperature induction during the heat shock response.

Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown. The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides. We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase. Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis. Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay. The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation. These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides.

The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response. We present experiments indicating a direct role for sigma 32 in controlling the heat shock response. Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32. Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32. These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.

The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.

We have analyzed some chemical properties of the sigma subunit of RNA polymerase from the sigma mutants: rpoD1 (Gross et al., 1978), rpoD2 (formerly known as alt-1) (Silverstone et al., 1972; Travers et al., 1978), and rpoD800 (Gross et al., 1979). Each of the three mutants is located at about 66 min on the E. coli genetic map and exhibits an alteration in the enzymatic properties of its sigma subunit. The tryptic peptides and isoelectric focusing behavior were analyzed for mutant and wild type sigma. A single, but different altered lysine tryptic peptide was observed for each mutant. No altered arginine tryptic peptides were observed. The rpoD800 mutant sigma showed an altered isoelectric point. These studies provide chemical evidence that the sigma polypeptide in all three mutants is altered and strongly support the conclusion that the mutations are in the structural gene for sigma.

We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62.5. Transcription from each promoter is heat-inducible in vivo, and each is recognized in vitro by RNA polymerase containing sigma 32, the sigma factor encoded by rpoH (htpR) but not by RNA polymerase containing sigma 70. We compared the sequences of the heat shock promoters and propose a consensus promoter sequence, having T-N-t-C-N-C-c-C-T-T-G-A-A in the -35 region and C-C-C-C-A-T-t-T-a in the -10 region. These sequences differ from the consensus sequence recognized by holoenzyme containing sigma 70, the major sigma in E. coli. We suggest that the accumulated consensus sequences of promoters recognized by alternate forms of holoenzyme are compatible with a model in which sigma recognizes only the -10 region of the promoter.

A transducing phage has been isolated with codes for the sigma subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage lambda vector that is capble of forming lyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the sigma gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and sigma (rpoD) whereas those containing the Sac I fragment complement only sigma. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/translation system suggest that a Sac I site separates the promoter for sigma from the sigma structural gene. The direction of transcription of sigma was determined to be clockwise with respect to the E. coli genetic map.

The htpR locus in Escherichia coli encodes a regulator of the heat shock response. Cells containing the htpR165 mutation are defective in the induction of synthesis of heat-shock proteins at high temperature. We show that these cells are also defective in degrading two proteins that are normally unstable in htpR+ cells. The proteolytic defect is manifest at both 30 degrees C and 42 degrees C. We used a marker rescue technique to map this defect to the htpR locus. Although both proteolytic substrates are partially stabilized in lon- strains, we argue that the defect in proteolysis exhibited by the htpR165 strain does not mimic the lon- state. The htpR165 strain synthesizes Lon at the normal rate at 30 degrees C and does not show the phenotypes of mucoidy and radiation sensitivity associated with lon- strains.

A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.

An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C. In the present study we characterize the nature of the inhibition. The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP). We inferred that the inhibition might be mediated by a decreased intracellular concentration of cAMP. Consistent with this inference, the rate of cAMP accumulation in mutant cells after a temperature upshift was depressed relative to that in wild-type cells. Glucose and chloramphenicol, two agents known to inhibit differentially beta-galactosidase mRNA synthesis, caused a similar inhibition in the rate of cAMP accumulation. Thus, three diverse stimuli, glucose, chloramphenicol, and a temperature-sensitive sigma mutation, appear to affect beta-galactosidase synthesis by regulating the synthesis of cAMP.

Escherichia coli K12 strains containing the mutant sigma allele rpoD800 are temperature-sensitive for growth. We have compared gene expression in isogenic rpoD+ and rpoD800 cells during steady-state growth and after temperature shift, in order to define the role of the sigma subunit in vivo. We have shown that sigma synthesis is regulated. After temperature shift-up, sigma behaves like other heat-shock proteins. The stimulation of sigma synthesis by heat shock is greater in mutant than in wild-type cells, possibly because the cell is responding to sigma limitation at high temperature by over-producing sigma. Mutant cells continue protein synthesis for a short time after shift-up and then shut off the synthesis of all proteins in response to the decreasing intracellular concentration of sigma. During the initial period of high protein synthesis, the relative expression of many proteins is changed in mutant cells. We argue that these changes are predominantly an indirect, rather than a direct effect of mutant sigma and are due to a change in the physiological state of mutant cells. Finally, we have shown that degradation of mutant sigma results in a decrease in synthesis of all major messenger RNA and stable RNA species. If other sigma factors are present in exponentially growing cells, they do not appear to be involved in a significant fraction of RNA synthesis.