RESUMO
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.
Assuntos
Escherichia coli/fisiologia , Resposta ao Choque Térmico , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/fisiologia , Retroalimentação , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/fisiologia , Matemática , Modelos Biológicos , Fator sigma/análise , Fator sigma/fisiologiaRESUMO
The anti-sequence, a portable element extending from +1 to +15 of the transcript, is sufficient to prevent promoter escape from a variety of strong final sigma70 promoters. We show here that this sequence does not function with even the strongest final sigma32 promoter. Moreover, a particular class of substitutions in final sigma70 that disrupt interaction between Region 2.2 of final sigma70 and a coiled-coiled motif in the beta'-subunit of RNA polymerase antagonizes the function of the anti-element. This same group of mutants prevents lambdaQ-mediated anti-termination at the lambdaP(R') promoter. At this promoter, interaction of final sigma70 with the non-template strand of the initial transcribed sequence (ITS) is required to promote the pause prerequisite for anti-termination. These mutants prevent pausing because they are defective in this recognition event. By analogy, we suggest that interaction of final sigma70 with the non-template strand of the anti-ITS is required for function of this portable element, thus explaining why neither final sigma32 nor the Region 2.2 final sigma70 mutants mediate anti-function. Support for the analogy with the lambdaP(R') promoter comes from preliminary experiments suggesting that the anti-ITS, like the lambdaP(R') ITS, is bipartite.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fator sigma/metabolismo , Sequência de Bases , DNA , RNA Polimerases Dirigidas por DNA/genética , Mutação , Fator sigma/genética , Moldes GenéticosRESUMO
The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Genes Reporter , Immunoblotting , Modelos Moleculares , Fragmentos de Peptídeos/genética , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator sigma/química , Fator sigma/genética , Transcrição GênicaRESUMO
For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Transcrição Gênica , Regulação Alostérica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Modelos Moleculares , Mutação , Desnaturação de Ácido Nucleico , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Fator sigma/químicaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Isopropiltiogalactosídeo/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Periplasma/metabolismo , Fator sigma/genética , Supressão Genética , Fatores de Transcrição/genéticaAssuntos
Distinções e Prêmios , Genética , Genética/história , História do Século XX , Mutagênese , Sociedades Médicas , Estados UnidosRESUMO
Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the sigma(70) subunit. A motA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five sigma(70) mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant sigma(70) proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The sigma(70) substitutions affected the 4.2 region, which binds the -35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. coli promoters. This defect could not be explained solely by a disruption in -35 recognition since similar results were obtained with extended -10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.
Assuntos
Bacteriófago T4/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Fator sigma/fisiologia , Fatores de Transcrição/genética , Proteínas Virais/genética , Proteínas Virais/fisiologia , Escherichia coli/crescimento & desenvolvimento , Mutação , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Proteínas de Bactérias/metabolismo , Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Genótipo , Cinética , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Plasmídeos , Dobramento de Proteína , Ribonuclease T1/química , Ribonuclease T1/metabolismo , Deleção de SequênciaRESUMO
The sigma subunit of eubacterial RNA polymerase is required throughout initiation, but how it communicates with core polymerase (alpha(2)betabeta') is poorly understood. The present work addresses the location and function of the interface of sigma with core. Our studies suggest that this interface is extensive as mutations in six conserved regions of sigma(70) hinder the ability of sigma to bind core. Direct binding of one of these regions to core can be demonstrated using a peptide-based approach. The same regions, and even equivalent residues, in sigma(32) and sigma(70) alter core interaction, suggesting that sigma(70) family members use homologous residues, at least in part, to interact with core. Finally, the regions of sigma that we identify perform specialized functions, suggesting that different portions of the interface perform discrete roles during transcription initiation.
Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Choque Térmico/química , RNA Polimerase I/química , Fator sigma/química , Fatores de Transcrição/química , Transcrição Gênica , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , RNA Polimerase I/metabolismo , Proteínas Recombinantes de Fusão/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40 , Cinética , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Citoplasma/metabolismo , Genes Reporter , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Mutagênese , Plasmídeos/metabolismo , Testes de Precipitina , Fator sigma/biossíntese , Transdução de Sinais , Temperatura , Fatores de Tempo , Fatores de Transcrição/biossínteseRESUMO
Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Estrutura Secundária de Proteína , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40 , Cinética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Fenótipo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoAssuntos
Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Sequência Consenso , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Filogenia , Fator sigma/química , Fator sigma/genéticaRESUMO
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.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Mutagênese , Proteínas Recombinantes/biossíntese , Fator sigma/antagonistas & inibidores , Fator sigma/genética , Supressão Genética , Temperatura , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Choque Térmico , Resposta ao Choque Térmico/genética , Proteínas Periplásmicas , Proteínas Quinases , Fator sigma/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/fisiologia , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos/fisiologia , Genes Supressores/genética , Lipoproteínas/genética , Fases de Leitura Aberta/genética , Fenótipo , Regulon/fisiologia , Análise de Sequência de DNA , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Fator sigma/genética , Fatores de Transcrição/genéticaRESUMO
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).
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Plasmídeos , Receptores Proteína Tirosina Quinases/fisiologia , Deleção de Sequência , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
Little is known about either the process of periplasmic protein folding or how information concerning the folding state in this compartment is communicated. We present evidence that SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, is involved in the maturation and assembly of LamB. LamB is a trimeric outer membrane porin for maltodextrins as well as the bacteriophage lambda receptor in Escherichia coli. We demonstrate that SurA is involved in the conversion of unfolded monomers into a newly identified intermediate in LamB assembly, which behaves as a folded monomer. The absence of SurA blocks the assembly pathway and leads to accumulation of species prior to the folded monomer. These species also accumulate when the stress sigma factor sigmaE is induced by LamB overexpression. We suggest that accumulation of species prior to the generation of folded monomer is a stress signal sensed by sigmaE.
Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Porinas/metabolismo , Receptores Virais/metabolismo , Escherichia coli/enzimologia , Peptidilprolil Isomerase , Dobramento de Proteína , Processamento de Proteína Pós-TraducionalRESUMO
The recent publication of the 2.6 A crystal structure of a portion of sigma70 provides insight into the role of sigma during transcription initiation. This high resolution picture unveils novel questions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Fator sigma/química , Transcrição Gênica , Cristalografia , Escherichia coli/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação ProteicaRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Genes Letais , Genes Recessivos , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Elongação Traducional da Cadeia Peptídica , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.