Isolation and characterization of mutations in region 1.2 of Escherichia coli sigma70.

Eubacterial RNA polymerase uses the sigma (sigma) subunit for recognition of and transcription initiation from promoter DNA sequences. One family of sigma factors includes those related to the primary sigma factor from Escherichia coli, sigma70. Members of the sigma70 family have four highly conserved domains, of which regions 2 to 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by sigma70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work has demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter-specific binding by holoenzyme. We generated random single amino acid substitutions targeted to region 1.2 of E. coli sigma70 as well as a deletion of region 1.2, and characterized the behaviour of the mutant sigma factors both in vivo and in vitro to investigate the function of region 1.2 during transcription initiation. In this study, we show that mutations in region 1.2 can affect promoter binding, open complex and initiated complex formation and the transition from abortive transcription to elongation.

Formation of intermediate transcription initiation complexes at pfliD and pflgM by sigma(28) RNA polymerase.

The sigma subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. Primary sigma factors are essential for growth, while alternative sigma factors are activated in response to various stimuli. Expression of class 3 genes during flagellum biosynthesis in Salmonella enterica serovar Typhimurium is dependent on the alternative sigma factor sigma(28). Previously, a novel mechanism of transcription initiation at the fliC promoter by sigma(28) holoenzyme was proposed. Here, we have characterized the mechanism of transcription initiation by a holoenzyme carrying sigma(28) at the fliD and flgM promoters to determine if the mechanism of initiation observed at pfliC is a general phenomenon for all sigma(28)-dependent promoters. Temperature-dependent footprinting demonstrated that promoter binding properties and low-temperature open complex formation are similar for pfliC, pfliD, and pflgM. However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at pflgM, while a sequential pattern of open complex formation occurs at pfliD. Open and initiated complexes formed by holoenzyme carrying sigma(28) are generally unstable to heparin challenge, with the exception of initiated complexes at pflgM, which are stable in the presence of nucleoside triphosphates.

Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.

The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein. Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R). To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes. We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters. Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences.

Effects of amino acid substitutions at conserved and acidic residues within region 1.1 of Escherichia coli sigma(70).

Amino acid substitutions in Escherichia coli sigma(70) were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

Transcription initiation at the flagellin promoter by RNA polymerase carrying sigma28 from Salmonella typhimurium.

The sigma subunit of RNA polymerase is a critical factor in positive control of transcription initiation. Primary sigma factors are essential proteins required for vegetative growth, whereas alternative sigma factors mediate transcription in response to various stimuli. Late gene expression during flagellum biosynthesis in Salmonella typhimurium is dependent upon an alternative sigma factor, sigma28, the product of the fliA gene. We have characterized the intermediate complexes formed by sigma28 holoenzyme on the pathway to open complex formation. Interactions with the promoter for the flagellin gene fliC were analyzed using DNase I and KMnO4 footprinting over a range of temperatures. We propose a model in which closed complexes are established in the upstream region of the promoter, including the -35 element, but with little significant contact in the -10 element or downstream regions of the promoter. An isomerization event extends the DNA contacts into the -10 element and the start site, with loss of the most distal upstream contacts accompanied by DNA melting to form open complexes. Melting occurs efficiently even at 16 degrees C. Once open complexes have formed, they are unstable to heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at rRNA promoters.

A mutation in region 1.1 of sigma70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme.

The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. It directs recognition of DNA sequences by holoenzyme (alpha2betabeta'sigma) and facilitates subsequent steps in the initiation pathway. The primary sigma factor from Escherichia coli, sigma70, has four regions that are conserved among members of the sigma70 family. Previous work has shown that region 1.1 modulates DNA binding by regions 2 and 4 when sigma is separated from the core subunits, and is required for efficient progression through the later steps of initiation in the context of holoenzyme. In this report, we show that an amino acid substitution at position 53 in region 1.1, which converts isoleucine to alanine (I53A), creates a sigma factor that associates with the core subunits to form holoenzyme, but the holoenzyme is severely deficient for promoter binding. The I53A phenotype can be suppressed by truncation of five amino acids from the C-terminus of sigma70. We propose that the behavior of sigma70-I53A is a consequence of impaired ability to undergo a critical conformational change upon binding to the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme.

Region 1 of sigma70 is required for efficient isomerization and initiation of transcription by Escherichia coli RNA polymerase.

The sigma (sigma) subunit of prokaryotic RNA polymerase is essential for promoter recognition. sigma Factor directs the RNA polymerase core subunits (alpha2beta beta') to the promoter consensus elements and thereby confers selectivity for transcription initiation. The N-terminal domain (region 1.1) of Escherichia coli sigma70 has been shown to inhibit DNA binding by the C-terminal DNA recognition domains. Since DNA recognition is the first step of transcription initiation, we predicted that the N-terminal domain of sigma70 may also influence the initiation of transcription by holoenzyme (alpha2beta beta'sigma). N-terminally truncated sigma70 derivatives were used to reconstitute holoenzyme, and transcription by the mutant holoenzymes was analyzed in vitro. Deletion of the first 75 to 100 amino acids of sigma70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA- strand-separated open complex, as well as a reduced efficiency of transition from the open complex to a ternary initiated complex. These effects were partially reversed by the addition of a polypeptide containing region 1.1 in trans. Therefore, region 1.1 not only modulates DNA binding but is important for efficient transcription initiation, once a closed complex has formed. A deletion of the first 133 amino acids, which removes regions 1.1 and 1.2, resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation.

Recognition of the -10 promoter sequence by a partial polypeptide of sigma70 in vitro.

Promoter recognition by RNA polymerase depends upon its ability to bind to specific DNA sequences. The sigma (sigma) subunit provides selectivity for transcription initiation by interacting with the -10 and -35 elements of promoter DNA. Suppressor mutations in sigma factor that compensate for specific "down" substitutions in the promoter have demonstrated that sigma factor recognizes certain base pairs of the promoter. Since these suppressors were only identified for changes at the -12 and -11 positions of the -10 element (TATAAT), the role of the other base pairs of this region in specifying recognition by sigma factor remained unclear. Using a partial polypeptide of sigma70 carrying regions 2-4, this report shows that the first three positions of the -10 element (-12, -11, -10) are important for sigma factor alone to recognize and bind to duplex DNA. The sigma polypeptide also binds to an "extended -10" promoter, even without a -35 element. A mismatch bubble from -10 to +3 is bound regardless of the sequence within the bubble, or the presence or absence of a -35 element. Unexpectedly, binding to a mismatch bubble that lacks a -35 hexamer is sensitive to the identity of the -11 position, but not the -12 position.

The role of the pro sequence of Bacillus subtilis sigmaK in controlling activity in transcription initiation.

The sigma (sigma) subunit of prokaryotic RNA polymerase is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of gene expression can therefore be achieved by modulating the activity of the sigma subunit. In Bacillus subtilis the mother cell-specific sporulation sigma factor, sigmaK, is synthesized as a precursor protein, pro-sigmaK, with a 20-amino acid pro sequence. This pro sequence renders sigmaK inactive for directing transcription of sigmaK-dependent genes in vivo until the pro sequence is proteolytically removed. To understand the role of the pro sequence in controlling sigmaK activity, we have constructed NH2-terminal truncations of pro-sigmaK and characterized their behavior in vitro at the gerE promoter. In this report we show that the pro sequence inactivates sigmaK by interfering with the ability of sigmaK to associate with the core subunits of polymerase and also influences the interactions between holoenzyme and promoter DNA. Additionally, removal of as few as 6 amino acids (pro-sigmaKDelta6) is sufficient to activate pro-sigmaK for DNA binding and transcription initiation. Surprisingly, pro-sigmaKDelta6 binds to DNA with higher affinity and stimulates transcription 30-fold more efficiently than sigmaK, under certain conditions.

The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing.

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.

Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase. Genetic studies have indicated that sigma itself interacts with DNA at the -10 and -35 promoter consensus sequences. Binding of Escherichia coli sigma 70 to DNA in vitro, however, can only be observed for truncated polypeptides lacking the amino-terminal amino acids. We have investigated the role of the amino terminus of E. coli sigma 70 in controlling DNA-binding ability. Deletion analysis indicates that amino acids within amino-terminal region 1.1 of sigma 70 inhibit DNA binding by the carboxy-terminal DNA-binding domains. Furthermore, inhibition of binding by the amino-terminal inhibitory domain of sigma 70 can be observed in trans. Likewise, the amino-terminal extensions of two alternative sigma-factors, E. coli sigma 32 and Bacillus subtilis sigma K, negatively affect the DNA binding activity of their carboxy-terminal domains. We propose that initiation of transcription is subject to modulation as a result of the composition and/or structure of the amino terminus of the sigma-subunit and that the sigma family of proteins belong to a larger class of intramolecularly regulated transcriptional effectors.

The role of the sigma subunit in promoter recognition by RNA polymerase.

Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase holoenzyme. Genetic and physiological studies have indicated that sigma interacts with promoter DNA sequences but biochemical analysis did not demonstrate DNA binding by the sigma subunit itself. We have investigated both the DNA binding properties and the regulation of DNA binding for several sigma factors using partial polypeptides. In this report we demonstrate that partial sigmas can bind to promoter DNA in the absence of the core subunits of RNA polymerase and the binding is regulated by an N-terminal inhibitory domain.

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Mutations in the ATP-binding domain of Escherichia coli rho factor affect transcription termination in vivo.

Five mutant rho proteins, representing alterations at three different locations in the Escherichia coli rho gene that affect ATP hydrolytic activity but not RNA binding, were examined in vivo for function at the rho-dependent IS2 and bacteriophage lambda tR1 terminators. The altered amino acids in rho are located at highly conserved residues near the beta 1 and beta 4 strands of the hydrophobic ATP-binding pocket that is structurally similar to the F1-type ATPases and adenylate kinase. The RNA-dependent ATPase activities of the mutant rho proteins were previously shown to range from undetectable to a twofold increase over wild-type rho in vitro. Analysis of these proteins within the environment of the cell confirmed that transcription termination in vivo is indeed related to the ability of rho factor to properly hydrolyze nucleoside triphosphates, as would be predicted from results in vitro. The relative efficiency of termination at lambda tR1, as judged by lambda N= plating efficiency and by suppression of polarity of IS2 upstream of galK, was closely linked to the level of RNA-dependent ATPase activity observed in vitro for each protein. Moreover, the termination efficiency of four of the altered rho proteins at IS2 and lambda tR1 in vivo corresponded directly to the effect of these mutations on rho function at the E. coli trp t' terminator in vitro. We conclude that determinations of rho function in vitro accurately reflect its behavior in intracellular termination events.

The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine.

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.

Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor.

We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase. Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine. The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination. Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding. These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase.

Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains.

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.

Transcription termination factor rho is an RNA-DNA helicase.

E. coli rho factor can unwind a short RNA-DNA duplex in vitro. The duplex is formed between a polylinker sequence at the 3' end of RNA derived from the rho-dependent terminator trp t' and the complementary sequence in a single-strand DNA molecule. Release of trp t' RNA from the duplex requires nucleoside triphosphate hydrolysis by rho's NTPase activity and is dependent on rho recognition of the RNA that is 5' to the RNA-DNA duplex region. The direction of helix unwinding appears to be 5' to 3' along the RNA molecule. These characteristics now account for how the RNA-binding and RNA-dependent NTP hydrolysis activities of rho may participate directly in transcription termination. Our results suggest that NTP hydrolysis is utilized to help unwind the RNA-DNA duplex at the 3' end of a nascent transcript, facilitating RNA release from the DNA template.