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Journal of Cell and Molecular Biology 2: 65-77, 2003. Haliç University, Printed in Turkey.

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A general view: Structure and function of the subunits of E. coli RNA polymerase Nihal Büyükuslu Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, 34280, F›nd›kzade, ‹stanbul, Turkey Received 9 June 2003; Accepted 23 June 2003

Abstract The DNA-dependent RNA polymerases are widespread throughout nature. E. coli RNA polymerase, one of the most well characterized polymerase, consists of two major forms, core enzyme with subunit stoichiometry of α2ββ' and holoenzyme which contains an additional σ subunit to core enzyme. E. coli RNA polymerase plays a central role in transcription. While the core enzyme catalyses the elongation and termination of transcription, to initiate core enzyme needs to combine with σ subunit. The three dimensional structure of this multimeric enzyme revealed a thumb-like projection. Using the electron microscope, Tichelar and Heel (1990) proposed a model that is in agreement with both β and β' together constituting a V-like structure and α dimer associates at the short ends, while σ is positioned within the concave side of the core, next to the dimer. In this review, the structure and related functions of the subunits of E. coli DNA-dependent RNA polymerase is presented based on several researches and reviews. Considering biochemical and genetic studies on the RNA polymerase of E. coli, a genetic walk on the subunits is summarized.

Key words: E. coli RNA polymerase, α subunit, β subunit, β' subunit, σ factors

Genel bak›fl: E. coli RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar› Özet DNA-ba¤›ml› RNA polimerazlar do¤ada yayg›n olarak bulunurlar. En iyi karakterize edilen polimerazlardan biri olan E. coli RNA polimeraz iki ana yap›dan oluflur, α2ββ' stokiyometrisi ile çekirdek enzim ve çekirdek enzime σ alt biriminin eklenmesi ile oluflan holoenzim. E. coli RNA polimeraz transkripsiyonda önemli bir rol oynar. Çekirdek enzim transkripsiyonun uzamas› ve sonlanmas›n› katalizlerken, transkripsiyonun bafllamas› için çekirdek enzime σ alt biriminin eklenmesi gerekmektedir. Bu çok altbirimli enzimin üç boyutlu yap›s› el ayas›na benzer bir yap› gösterir. Elektron mikroskobu kullanarak Tichelar ve Heel (1990)’in önerdikleri modele göre β ve β' alt birimleri birlikte V fleklinde yap› oluflturmakta ve α dimeri k›sa uçlarla birleflmektedir, σ ise çekirde¤in konkav k›sm›nda dimere komflu olarak yer almaktad›r. Bu derlemede çeflitli araflt›rmalar ve derlemeler baz al›narak E. coli DNA-ba¤›ml› RNA polimeraz›n alt birimlerinin yap›lar› ve fonksiyonlar› sunulmufltur. E. coli RNA polimeraz›n üzerinde yap›lan biyokimyasal ve genetik incelemelere dayan›larak alt birimleri üzerinde genetik bir yürüyüfl özetlenmifltir.

Anahtar sözcükler: E. coli RNA polimeraz, α alt birimi, β alt birimi, β' alt birimi, σ faktörleri

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Structure and function of DNA-dependent RNA polymerase of E. coli Transcription in wide range organisms uses a homologous family of multisubunit DNA-dependent RNA polymerases. The complex, multimeric DNAdependent RNA polymerases are highly conserved throughout nature, suggesting a common evolutionary origin. A considerable body of research has allowed the identification of potential functional regions within several of the subunits. The DNA-dependent RNA polymerase plays a central role in transcription. During transcription in E. coli, RNA polymerase is involved following steps: RNA polymerase i) locates specific promoter sequences in the DNA template; ii) melts a small region containing the transcription start site; iii) initiates RNA synthesis; iv) elongates the transcript, and finally v) terminates and releases the RNA product. Each step in this process is regulated by interaction between the polymerase, the DNA, the nascent RNA and some regulatory proteins and ligands. RNA polymerase consists of two major forms, core enzyme which elongates and terminates transcription and holoenzyme, which contains an additional σ subunit responsible for the initiation of transcription. The core enzyme consists of two α subunits, one β and one β' subunit with relative molecular masses of 36.511; 150.616; 155.159 kD, respectively. The ω subunit (10.237 kD) is also associated with RNA polymerase. The subunits of RNA polymerase are shown in Table 1.

Figure 1: Three-dimensional structure of E. coli RNA holo-polymerase by electron microscopy (Darst et al, 1989).

Three-dimensional structure of E. coli RNA polymerase by electron microscopy revealed a thumblike projection, similar to the active site cleft of DNA polymerase I (Figure 1) (Darst et al., 1989). Polyakov et al. (1995) supported the thumb-like projection surrounding a groove or channel about 25Å in diameter. The thumb of E. coli RNA polymerase holoenzyme defines a deep but open groove on the surface of the enzyme while in E. coli core RNA polymerase the thumb forms part of a ring that completely enclose the channel. A recent study (Vassylyev et al., 2002) reported the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6Å resolution. In the structure, two amino-terminal domains of the σ subunit form a V-shaped structure near the opening of

Table 1: The subunits of RNA polymerase of E. coli. Subunit

Size aa

Size kD

Gene

Function

Alpha (α)

329

36.511

rpoA

Beta (β)

1342

150.616

rpoB

Beta'(β') Sigma (σ) Omega (ω)

1407 613 91

155.159 70.263 10.237

rpoC rpoD rpoZ

Required for assembly of the enzyme; interacts with some regulatory proteins; also involved in catalysis Catalysis of RNA synthesis (initiation and elongation); recognition of terminators; binding of substrate ribonucleoside 5'-triphosphates; binding of product RNA; stringent control; autogeneous regulation of ββ' synthesis; binding of rifampicin and streptolydigin Binds to the DNA template; binds to sigma subunits Promotion of core enzyme maturation; recognition of regular promoters Required to restore denatured RNA polymerase in vitro to its fully functional form.

E. coli RNA polymerase

the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of σ is near the outlet of the RNA-exit channel, about 57Å from the Nterminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation. The subunits of E. coli RNA polymerase

The α subunit The α subunit of E. coli DNA-dependent RNA polymerase is encoded by the rpoA gene and is composed of 329 amino acid residues. The α subunit maps within the large cluster of ribosomal genes located at the 72-minute region of the E. coli chromosome. Genetic and biochemical studies indicate the α subunit carries out three critical functions; subunit assembly, promoter recognition by direct sequence-specific protein-DNA interaction, transcription activation by a set of activator proteins. Limited proteolysis experiments showed that the α subunit consists of N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. Although the α N-terminal region contains determinants for interaction with the remainder of RNA polymerase and the α C-terminal region contains determinants for interaction with transcription activator proteins (Hayward et al., 1991; Igarashi et al., 1991; Igarashi and Ishihama, 1991) the C-terminal 85 amino acids of RNA polymerase a constitute an independently folded domain that is shown to be capable of dimerisation and sequencespecific DNA binding. Furthermore, a C-terminal deleted α mutant consisting of the N-terminal 235 amino acid residues retains the ability to form α mutant core enzyme both in vitro (Igarashi et al., 1991; Igarashi and Ishihama, 1991) and in vivo (Hayward et al., 1991). For detailed mapping of the N-terminal assembly domain, Kimura et al. (1994) made a set of N-terminal and internal mutants and found that the minimum region of the α subunit required for core enzyme assembly is located between residues 21 and 235. The temperature-sensitive mutant, rpoA112 at

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position 45, blocks RNA polymerase assembly (Igarashi et al., 1990; Ishihama et al., 1980; Kawakami and Ishihama, 1980). In support of this, Thomas and Glass (1991) have found that expression of a truncated a derivative comprising the N-terminal 230 amino acids results in complementation of the Mel phenotype of the rpoA341 mutant confirming that this region of α contains all that is necessary for assembly in vivo. The substituted deletion derivatives of the same region were shown to be capable of complementing the Nterminal αTs mutation suggesting that the presence but not necessarily the nature of the sequences C-terminal to residue 287 serve as a molecular ‘scaffold’ facilitating the formation of the correct conformation without being intrinsically required for function per se (Zou C. Personal communication) Recent functional mapping within this assembly domain by making a series of insertion mutants having two extra amino acids at every 20 residues, led to a proposal for the functional organization of the Nterminal assembly domain of the α subunit. The region around residue 80 is involved in binding both β and β': the region including residues 180 and 200 plays a role in β' binding and in dimerisation, more than one contact site widely distributed within this assembly domain are involved or alternatively multiple sites form a single contact surface (Kimura and Ishihama, 1995). Indeed, a region near residue 80 exists with a high content of hydrophobic amino acid residues that appears to be involved in binding both β and β'. Furthermore, mutations near the C-terminal proximal region in the assembly domain affect β' binding. Although the α subunit has been assigned a role mainly in the assembly of the multisubunit complex, much evidence suggests that α is also involved in interactions with transcriptional regulators and plays a central role in the resulting control of polymerase activity. In E. coli RNA polymerase, the C-terminal region of the α subunit is claimed to be the contact site for the UP element of the rrnB P1 promoter that is required for the transcription activation of the target ribosomal RNA gene. Transcription and DNase I footprinting results with RNA polymerases containing C-terminal deletions in the α subunit suggest that UPlike elements which interact with the α subunit might play a role in transcription at promoters that have sequences rich in (A+T) in upstream promoter elements (Ross et al., 1993). An analysis of the Cterminal domain of the E. coli RNA polymerase α subunit (αCTD) by nuclear magnetic resonance

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spectroscopy showed that the structure of αCTD is compactly folded and comprised of four helices and two long loops at the ends of the domain. A chemical shift perturbation experiment was performed to observe which residues of αCTD are involved in the interaction with UP promoter elements. The residues affected by perturbation were attributable to amides of most of the residues from Glu261 to Ile275 and from Thr292 to Ile303 that are located in helix 1, the Nterminal end of the helix 4, and the preceding loop. The C-terminal region of the α subunit is responsible for contact with cis-acting UP element as well as with trans-acting transcription factors. One of the well characterized transcription factors, cAMPCRP, binds to specific (22bp) sites on DNA, the position of the site(s) depending on the particular promoters. The cAMP receptor protein, CRP, controls the initiation of transcription of several genes especially those involved in carbon-source utilization. Activation of transcription by the upstream CRP molecule is blocked by the HL159 substitution, suggesting that the upstream-bound CRP makes a direct contact with RNA polymerase. Footprinting experiments indicated that RNA polymerase contacts the promoter DNA between the two CRP-binding sites, most likely due to interactions involving the C-terminal part of the α subunit (Attey et al., 1994). Although many CRP-dependent promoters carry a single CRPbinding site, centered around –40, –60 or –70, a number of promoters carry multiple CRP-binding sites. In order to accommodate direct contacts between both CRP dimers and the two α subunits in ternary complexes at the ML1 promoter, Busby et al. (1994) proposed a model that the α subunits are sandwiched between CRP and RNA polymerase. Since the α dimer is able to bind directly to DNA (Ross et al., 1993) it seems probable that the α subunits are responsible for the upstream RNA polymerase contacts and that the α dimer bridges the two CRP dimers. In contrast, at class II promoters, α binds just upstream of the CRP dimer and makes contact with via activating region I. Although the CRP dimer contains an activating region I in each subunit, it is only the activating region I in the upstream of the CRP dimer that makes contact with RNA polymerase during transcription initiation. These results suggest that α makes contact with the upstream subunit of the CRP dimer whilst the downstream subunit is likely to make alternative contacts with other parts of RNA polymerase (Attey et al., 1994).

The ß subunit The second largest subunit of E. coli RNA polymerase is composed of 1342 amino acids and is highly conserved throughout evolution (Sweetser et al., 1987; Iwabe et al., 1991; Ovchinnikov et al., 1981). The β subunit alone has no apparent function like the other subunits. When assembled into the RNA polymerase complex, β subunit has been shown to be involved in most of the catalytic functions of RNA polymerase, including nucleotide binding (Jin and Gross, 1991; Mustaev et al., 1991), transcription initiation, elongation and termination (Mustaev et al., 1991; Jin and Gross, 1988; Kashlev et al., 1990; Landick et al., 1990; Lee and Goldfarb 1991; Jin 1994), interactions with both the σ subunit (Glass et al., 1986;1988) and the NusA proteins (Jin and Gross, 1988; Sparkowski and Das, 1992). Mutations conferring resistance to rifampicin define several clustered residues in the central and amino terminal parts of the β subunit (Lisitsyn et al., 1984; Severinov et al., 1993; Ovchinnikov et al., 1981; 1983; Jin and Gross 1988). Sequence similarities among the subunits of different organisms implied three main domains; Nterminal domain, middle domain and C-terminal domain, and two dispensable regions centered around residues 300 and 1000. The conserved regions are thought to be important for function and structure of β subunit and the homology of these regions among organisms reveal the evolution of genetic information flow. Deletions in the N-terminal region of subunit of E. coli RNA polymerase between the residues 166-328 and 186-433 showed no obvious effect on function in vitro, suggesting that this region is dispensable for minimal function. The ∆(166-328) alteration was also found to be non-lethal in vivo (Severinov et al., 1994). A 69-residue segment between the residues 339-409 of β subunit is widespread among prokaryotes indicating that this region might play a structural or functional role. Indeed, a recent study showed that the β subunit residues 186-433 and 436-445 are commonly used by σ54 and σ70 RNA polymerase holoenzyme for open promoter complex formation (Wigneshweraraj et al., 2002). In E. coli this region also contains the paf32 alteration (Severinov et al., 1994) due to a contact site with the Alc protein, a site-specific termination factor encoded by bacteriophage T4 that acts as a block to the transcription of host genes (Kashlev et al., 1993). In support of this, Landick et al. (1990) identified a series

E. coli RNA polymerase

of substitutions in that region that affects transcription termination in vivo. All known mutations resulting in rifampicin resistance map in the β subunit (Halling et al., 1978; Miller et al., 1994) and lie between residues 512-573 (Kashlev et al., 1990; Landick et al., 1990; Jin and Gross, 1988; Lisitsyn et al., 1984; Severinov et al., 1993). Because the rifampicin resistant mutants in both regions showed the same phenotypes in vivo and in vitro, Severinov et al. (1993) have suggested that these two regions perform a common catalytic function in the core enzyme. Furthermore, cross-linking studies with initiating substrate analogues implied that the rifampicin region in the middle domain of β (residue 515-540) is placed a few angstroms away from Lys1065 and His1237 of the C-terminal domain despite their separation in the linear sequence of β by more than 500 amino acids (Severinov et al., 1995). The intragenic suppression data also supported the idea that β subunit residues 529, 1237, 564 and 142 might interact with each other in the folded ternary structure of the enzyme. Part of the conserved domain 3 of σ70 and the positions –3 and –4 of the template DNA strand are in contact with γ-phosphate of the initiating nucleotide indicating a close proximity to catalytic center of the enzyme (Severinov et al., 1994; Mustaev et al., 1994). Footprinting experiments revealed contact between the Lys1065 in the β subunit and 5' end of the nascent RNA chain (Krummel and Chamberlin, 1992; Mustaev et al., 1993). The rpoB gene of E. coli RNA polymerase is involved in streptolydigin resistance as well as in rifampicin resistance. Four contiguous amino acids have been mapped as the target for streptolydigin resistance. Although this region (543-546) lies in close proximity to the Rif cluster, there does not appear to be any functional overlap in terms of drug resistance between the RifR and StlR regions since StlR mutants are not altered in their sensitivity to rifampicin (Heisler et al., 1993). Moreover, mutations at amino acids 543546 do not appear to affect the functioning of RNA polymerase in vivo. A histidine-tagged mutation at amino acid 540 was able to transcribe in the presence of Stl. These results suggest that the StlR region is dispensable for RNA polymerase activity and is probably looped out or surface exposed and there is a structural effect between the StlR region in β and the nearby catalytic center of the subunit. A fine mapping of amber mutations in the β gene by virtue of the unique MaeI restriction site created by this subset of nonsense mutations revealed that the deletion of 31

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amino acids between 618-649 is assembled into a holoenzyme form capable of transcriptional initiation in vivo (Buyukuslu et al., 1997). Although this region common to the eubacterial chloroplast subgroup of β homologous it may not responsible for assembly and initiation of transcription. Another region that is dispensable for normal β function is placed at residues 940-1040 (Borukhov et al., 1991; Severinov et al., 1992). This region is also missing from B. subtilis, the homologous RNA polymerase subunits from mycobacteri, chloroplast, eukaryotes and archaebacteria (Miller et al., 1994; Honore et al., 1993; Borukhov et al., 1991). Nene and Glass (1984) identified a part of the C-terminal region that is largely inessential, and removal of 62 residues (965-1143) resulted in a wild type phenotype other than a slightly slower growth on minimal medium. In vitro reconstitution studies (Glass et al., 1986; 1988) suggest that the region between the residues 1180-1339 is necessary for holoenzyme formation. Furthermore, after deletion of the C-terminal region of E. coli β retained the ability to associate with β' and α subunits indicating that this region is important in binding σ. A recent study using trans-dominant mutations in the 3' terminal region of the rpoB gene defined highly conserved essential GEME motif. The in vitro properties of the trans dominant-negative mutants in the GEME motif (1271→1274) emphasize the functional significance of this region, and contain residues that are in close vicinity of the active centre of the enzyme directly involved in catalysis (Cromie et al., 1999). Supporting the functionality of GEME motif, alanine substitution of three of the four GEME residues (i.e. RFGEME→AFGAAA) greatly reduced the affinity of RNA polymerase for substrates, without affecting promoter binding or the maximal enzymatic rate (Polyakov et al., 1999). Moreover, intragenic suppression of trans-dominant lethal substitutions in the GEME motif resulted that the GEME and HLVDDK (1237→1242) regions are present as α-helices in holoenzyme, and that functional cooperativity is through one particular face of each helix (Malik et al., 1999). In fact, the region 1243→1304 has been shown to contact the nascent RNA in the elongation complex (Gusarov and Nudler, 1999) indicating a possible catalytic site for both β and β' subunits.

The ß' subunit The β' subunit of E. coli RNA polymerase is composed

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of 1407 amino acid residues and is encoded by rpoC gene (Ovchinnikov et al., 1982; Squires et al., 1981). The early studies suggested that the β' subunit is essential for cell growth in the presence of a temperature sensitive lethal rpoC mutant (Panny et al., 1974). Due to its positive charge, the largest subunit can bind nonspecifically to DNA in the absence of the other subunits (Zillig et al., 1970) and is also able to bind other polyanionic molecules, including heparin (Fukuda and Ishihama, 1974). Some of β' mutations has been found to cause defects in the assembly of RNA polymerase (Toketo and Ishihama, 1976). Recently, Naryshkina et al. (2001) indicated that the β' subunit of E. coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin. Recent studies provided new insight into the structural and functional characteristics of β' subunit, especially its role in termination transcription. To identify regions of the β' subunit of RNA polymerase that are potentially involved in transcription elongation and termination Weilbaecher et al. (1994) have characterised amino acid substitutions in the β' subunit that alter expression of the genes preceded by terminators in vivo. Based on the location and properties of these substitutions they suggested a hypothesis that some intervals in β and β' defined by termination-altering substitutions may contact the DNA template or RNA transcript near the active site (residues 490-570 in β, 300-400 and 700800 in β' ). Among the conserved regions of β' subunit, a strongly basic region is involved in zinc-binding. Deletions in this N-terminal region of the β' subunit gave rise a lethal phenotype. A study by Clerget et al. (1995) indicated that deletions in the zinc-binding domain could block factor-independent antitermination and increase termination in coliphage HK022 DNA, indicating a nucleic acid-polymerase contact for termination. A region of the β' subunit shows similarity to E. coli DNA polymerase I. Substitutions in this region identified a portion of β' that contacts the DNA template, RNA transcript, or both, immediately upstream from the active site in RNA polymerase (Weilbaecher et al., 1994), although a conserved region which is the location of all known amanitinresistance substitutions in Pol II may fold in two distinct segments that are separated by a surfaceexposed region. These segments could interact to form

a functional site on the enzyme. Substitutions in region 4 altered the ratio of transcripts initiated at two adjacent start sites of a Pol II promoter (Hekmatpanat and Young, 1991). Moreover, cross-link mapping showed an interaction between E. coli RNA polymerase and 8-azido AMP at the 3' end of the nascent transcript occurring within the region spanning Met-932 and Trp-1020 of the β' subunit (Borukhov et al., 1991). Single substitutions downstream of this region exhibited a strong effect in vitro at some terminators indicating a direct effect on termination, or a retention of residual transcription factors during purification. In addition, the involvement of the β subunit in transcription termination has been suggested based on the isolation of rpoC mutations that suppress rho201 (Jin and Gross, 1989). An Arg-rich domain of NusA containing nusA1 and nusA11 has also been reported to interact with a regulatory component for transcription termination involving the β' subunit (Ito et al., 1991). Epitope mapping of monoclonal antibodies directed against the β' subunit of E. coli RNA polymerase indicated that the region 817-876 may be important in enzyme assembly or subunit-subunit interaction and the region located between amino acids 1047-1093 may be involved in the catalytic function of RNA polymerase (Luo and Krakow, 1992). A G to C transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the β' subunit affects chromosomal replication control in E. coli (Petersen and Flemming, 1991). 190 amino acid-long region centered around position 1050 of the 1407 amino acid-long β' subunit of E. coli RNA polymerase is absent from homologues in eukaryotes, archaea, and many bacteria. Moreover, in chloroplasts the corresponding region can be more than 900 amino acids long. The deletion mutagenesis of this hypervariable region revealed that long deletions mimicking β' of Gram positive bacteria failed to assemble into RNA polymerase. Short, 40-60 amino acids-long deletions spanning β' residues 9411130 assembled into active RNA polymerase in vitro. It is proposed that mutations in functionally dispensable region of β' inhibit transcript cleavage and elongation by distorting the flanking conserved segment in the active center supporting the previous results (Zakharova et al., 2003). Substitutions in the C-terminal region of β' appear to affect elongation of transcription. Furthermore, one conditional lethal and two suppressor substitutions in the yeast PolII homologue of β' occur in the conserved

E. coli RNA polymerase

region, supporting the idea that they may affect transcription in the same way (Weilbaecher et al., 1994).

The σ subunit In eubacteria, initiation of transcription is mediated by the σ subunit of RNA polymerase. However, free σ70 is not able to bind specifically at promoter DNA sites (Wellmann and Meares, 1991) due to the autoinhibition of σ70 DNA binding activity by the Nterminal domain of σ70 (Dombroski et al., 1992;1993). Although σ70, the major σ factor of E. coli, is required for initiation at most promoters, alternative σ factors are also responsible for transcription from other classes of promoters. Based upon characterisation, identification and sequence analysis, σ factors can be classified in two broad classes. One family is similar to the originally identified E. coli σ70 subunit, the other is similar to the 54 kD E. coli σ subunit. Before beginning a comparative analysis of σ families it is appropriate to discuss the structure and function of the σ factor. Sequence alignments of the σ family of proteins show similarities suggestive of common ancestry to reflect the various functional roles of σ factors in the cell (Gribskov and Burges, 1986; Stragier et al., 1985). In an expanded alignment of σ, four regions of high conservation have been identified (Lonetto et al., 1992). Regions 2 and 4 are the most conserved regions and tend to be very basic. Regions 1 and 3 exhibit lower conservation and are acidic. Considering the alignment of the σ70 family of sequences, it is possible to divide σ factors into three groups. The first group is comprised of primary σ factors that exhibit very high similarity and are responsible for most RNA synthesis in exponentially growing cells. Group two proteins are quite similar in sequence to the primary σ, but are dispensable for cell growth. The third group contains the alternative σs, which is responsible for transcription of specific regulons. Analysis of conserved regions of σ70 factors indicated that the four conserved regions could be further divided into subregions. The N-terminal regions of the major σ factors from various bacteria contain approximately 30% identical amino acid, these can be separated into two subregions: 1.1 and 1.2. Region 1.1 is rather poorly conserved and is present only in primary σs and E. coli Sig(38). Region 1.2 has several residues that are conserved among all primary

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and alternative σs expect M. xanthus SigB and S. typhimurium FliA. Although the role of region 1.2 has not been ascribed yet, the high conservation and a 14 amino acid deletion at position 330 that reduces the stability of σ70 at both low and high temperature (Hu and Gross, 1983) imply that this region is structurally and functionally important. Region 2 has been divided into four subregions; Region 2.1 and 2.3 are rich in aromatic amino acid residues. A deletion in region 2.1 reduces the binding of E. coli σ70 to core RNA polymerase suggesting that this region of σ involved in binding to core enzyme (Lesley and Burgess, 1989). There is evidence that regions 2.1 and 2.3 are involved in promoter unwinding in the region from –9 to +3 during formation of the open promoter complex (Siebenlist et al., 1980; Kirkegaard et al., 1983). Supporting this idea, a mutation in region 2.3 of SigE, and in the same region of B. subtilis SigA, interrupt the process of DNA strand separation during transcription initiation (Jones and Moran, 1992). Region 2.2 is the most conserved region among all groups of σ. Region 2.4 is highly conserved among primary σ, and implicated in recognition of the –10 region, while a helix-turn-helix motif located in subregion 4.2 of σ is involved in recognising the –35 consensus sequence (Daniels et al., 1990; Khan and Ditta, 1991; Siegele et al., 1989; Tatti et al., 1991; Waldburger et al., 1990; Zuber et al., 1989). More recently it was shown that conformation of conserved domains of σ70 (region 1, 2.4 and 4.2) was affected by the core enzyme (Callaci et al., 1998). Supporting that luminescence resonance energy transfer measurements showed that E. coli RNA polymerase induced major changes in σ70 involves a movement of the conserved region 1 by~20Å and the conserved region 4.2 by~15Å with respect to conserved region 2. This movement of DNA-binding domains of σ70 is thought to be an important mechanism by which the ability of σ70 to recognize promoter DNA is regulated (Callaci et al., 1999). Region 3 is divided into two subregions; 3.1 is more conserved with a weak resemblance to the helix-turnhelix and 3.2 is largely acidic and highly conserved among group 1 σs, but is weakly conserved among the group 3 σs. A 25 amino acid deletion in region 3.2 of E. coli σ causes an affinity reduction of this mutant protein to core enzyme indicating a possible secondary core-binding site (Lonetto et al., 1992). Region 4 consists of two subregions; 4.1 is an amphipatic α-helix and 4.2 is highly conserved among

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the primary σ factors and has a sequence similarity to the HTH-DNA binding motif (Brennan and Matthews, 1989; Gribskov and Burgess, 1986; Helmann and Chamberlin, 1988; Straiger et al., 1989). A spacer region of variable length and sequence lies between the two subregions. It appears likely that the conserved HTH motif in subregion 4.2 may directly contact the –35 promoter region. Although two mutations, R584C and R588H, in this region of the E. coli σ70 factor have been implicated in recognition of the –35 promoter sequence, a set of C-truncated σ70 lacking the –35 recognition domain can activate class II promoters (the pstS and P1gal), indicating that necessary contact or activation sites for the two activators lie at different positions in a segment of σ70 extending from region 3.2 to the upstream helix within region 4.2 (Kumar et al., 1994; Makino et al., 1993). Further evidence to support this suggestion came from the study by Jin et al., (1995). Using protein-protein photo-crosslinking, they showed that the CRP site for galP1, a class 2 promoter, contacts with amino acids 530-539 of σ70 region. All RNA polymerase holoenzymes containing σ70related σs appear to function in a similar manner. However, σ54 (σN), encoded by rpoN, is quite distinct, both structurally and functionally, from the σ70 family. Among 17 sequenced rpoN genes, there is a high degree of conservation in the protein primary sequence, and three distinct regions can be identified (Merrick et al., 1987). Region I is the N-terminal region is suggested to be α-helical; it is rich in leucine and glutamine residues. Region II varies between 60 to 110 residues in length, with essentially no sequence conservation. The C-terminal region III is highly conserved and contains two motifs; a potential helixturn-helix (HTH) motif and the ‘RpoN box’. σ54 and σ70 participate in different transcription mechanisms but bind the same core RNA polymerase (Gralla, 1991). Recent considerations suggest that σ54 should contain a minimum of two different elements

for recognition of the –24 and –12 promoter regions. C-terminal deletions near to the HTH motif abolished DNA binding (Sasse-Dwight and Gralla, 1990). Indeed, specific amino acid substitutions in this region showed a similar phenotype (Coppard and Merrick, 1991). Merrick and Chambers (1992) found a specific HTH mutation in K. pneumoniae σN (R363K) suppressed down mutations at position –13 in the glnAp2 promoter, suggesting that the conserved HTH region is involved in recognition of promoter-proximal sequences. Physical studies indicate that amino acids in a region from Asn312 to Arg345 in K. pneumoniae σN, just upstream of the HTH, crosslink to DNA in the presence or absence of core enzyme (Cannon et al., 1994). In contrast to the σ70 family, activation of Eσ54 is not dependent on the presence of the C-terminal domain (Lee et al., 1993). Transcription by Eσ54 appears to be controlled by a mechanism that requires the use of an activator protein and ATP to catalyze formation of open complexes (Popham et al., 1989). Deletions in both region I and region III prevent opencomplex formation. During open-complex formation, region II has been proposed to play a role in triggering conformational changes for DNA melting (SasseDwight and Gralla, 1990). The importance of σS has been increasingly recognised in recent years. This sigma factor appears only as cells enter the stationary phase of growth. It is responsible for transcription of all of the genes whose products are required during stationary phase (Ishihama, 2000). Two families of σ factors have been characterised in detail. A number of different σ factors belonging to sigma families are presented in Table 2 according to the origin, genetic locus, and presumed function.

The ω subunit The ω subunit, encoded rpoZ, is associated with both

Table 2: The E. coli sigma factors Sigma factor

Gene

Function

σ70 σ54 σ32 σS σF σFecI

rpoD RpoN (ntrA, glnF) RpoH RpoS RpoF fecI

Housekeeping function Nitrogen-regulated gene transcription Heat-shock gene transcription Gene expression in stationary phase cells Expression of flagellar operons Regulates the fec genes for iron dicitrate transport

E. coli RNA polymerase

core and holoenzyme (Burgess, 1969). The omega subunit was for many years considered a curiosity since no function could be ascribed to it. Therefore, the available literature on the ω subunit as regards to its structure and function is rare. The ω subunit is not required for the function of the transcriptional apparatus both in vivo and in vitro. Strain lacking ω, that is, a rpoZ null mutant is viable, suggesting a nonessential nature of the protein or that there might also be a redundancy in function. The only known phenotype ascribed to the rpoZ null mutant is a slower growth time (Mukherjee et al., 1999). However, it is now known that omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form. It may function by binding simultaneously to the N-terminus and C-terminus of the β' subunit. The omega subunit is a part of the Thermus aquaticus enzyme whose structure was recently determined (Murakami et al., 2002). A study by Mukharjee and Chatterji (1999) showed that ω-less holoenzyme has lesser affinity towards the DNA template and external addition of ω destabilizes the open complex for both the wild-type and ω-less enzyme. The ω-less core enzyme interacts with the σ70 subunit to expose the –35 recognition domain (domain 4·2) unlike that observed in the wild-type interaction. Thus the absence of the ω subunit leads to the formation of an enzyme which has altered DNA binding and σ70 binding properties. Circular dichroic measurements also indicate a major conformational alteration of both holo and core RNA polymerase in the presence and absence of the ω subunit. References Attey A, Belyaeva T, Savery N, Hoggett J, Fujita N, Ishihama A and Busby S. Interactions between the cyclic AMP receptor protein and the α subunit of RNA polymerase at the E. coli galactose operon P1 promoter. Nucl Acid Res. 22: 4375-4380, 1994. Borukhov S, Lee J and Goldfarb A. Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase. J Biol Chem. 266: 23932-23935, 1991. Brennan RT and Matthews BW. The helix-turn-helix DNA binding motif. J Biol Chem. 264: 1903-1906, 1989. Burgess RR. Separation and characterization of the subunits of RNA polymerase. J Biol Chem. 244: 2168-2176, 1969. Busby S, West D, Lawes M, Webster C, Ishihama A and Kolb A. Transcription activation by the E. coli cyclic-

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amp protein-receptors bound in tandem at promoters can interact synergistically. J Mol Biol. 241: 341-352, 1994. Büyükuslu N, Trigwell SM, Lim PP, Fujita N, Ishihama A, Ralphs N and Glass RE. Physical mapping of a collection of MaeI-generating mutations in the β gene of E. coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation. Genes and Function 1: 119-129, 1997. Callaci S, Heyduk E and Heyduk T. Conformational changes of E. coli RNA polymerase σ70 factor induced by binding to the core enzyme. J Biol Chem. 273: 329995-33001, 1998. Callaci S, Heyduk E and Heyduk T. Core RNA polymerase from E. coli induces a major change in the domain arrangement of the σ70 subunit. Molecular Cell. 3: 229238, 1999. Cannon W, Claveriemartin F, Austin S and Buck M. Identification of a DNA-contacting surface in the transcription factor σ54. Mol Microbiol. 11: 227-236, 1994. Clerget M, Jin DJ and Weisber RA. A zinc-binding region in the β' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022. J Mol Biol. 248: 768-780, 1995. Coppard JR and Merrick MJ. Casette mutagenesis implicates a helix-turn-helix motif in promoter recognition by the novel RNA-polymerase σ-factor σ54. Mol Microbiol. 5: 1309-1317, 1991. Cromie K, Ahmad K, Malik T, Büyükuslu N and Glass RE. Trans-dominant mutations in the 3'-terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif. Genes to Cells. 4: 145-159, 1999. Daniels D, Zuber R and Losick R. Two amino acids in an RNA polymerase σ factor involved in the recognition of adjacent base pairs in the –10 region of a cognate promoter. P roc Nat Acad Sci. USA 87: 8075-8079, 1990. Darst SA, Edwards AM and Kornberg RD. Threedimensional structure of E. coli RNA polymerase holoenzyme determined by electron crystallography. Nature. 340: 730-732, 1989. Dombroski AJ, Walter WA, Record MT, Jr. Siegele DA and Gross CA. Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Cell. 70: 501512, 1992. Dombroski AJ, Walter WA and Gross CA. Amino-terminal amino acids modulate sigma-factor DNA-binding activity. Genes Dev. 7: 2446-2455, 1993. Fukuda R and Ishihama A. Subunits of RNA polymerase in function and structure. V. Maturation in vitro of core enzyme from E. coli. J Mol Biol. 87: 523-540, 1974. Glass RE, Honda A and Ishihama A. Genetic studies on the β subunit of E. coli RNA polymerase. IX The role of the carboxy-terminus in enzyme assembly. Mol Gen Genet.

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203: 492-495, 1986. Glass RE, Ralphs NT, Fujita N and Ishihama A. Assembly of amber fragments of the β subunit of E. coli RNA polymerase. Eur J Biochem. 176: 403-407, 1988. Gralla JD. Transcriptional control-lessons from an E. coli promoter data base. Cell. 66: 415-418, 1991. Gribskov M and Burgess RR. σ factors from E. coli, B. subtilis, phage SP01 and phage T4 are homologous proteins. Nucl Acid Res. 14: 6745-6763, 1986. Gusarov I and Nudler E. The mechanism of intrinsic transcription termination. Mol Cell. 3: 495-504, 1999. Halling SM, Burtis KC and Doi RH. β' subunit of bacterial RNA polymerase is responsible for streptolydigin resistance in Bacillus subtilis. Nature. 272: 827-842, 1978. Hayward RS, Igarashi K and Ishihama A. Functional specialization within the α subunit of E. coli RNA polymerase. J Mol Biol. 221: 23-29, 1991. Heisler LM, Suzuki H, Landick R and Gross CA. Four contiguous amino-acids define the target for streptolydigin resistance in the β subunit of E. coli RNA polymerase. J Biol Chem. 268: 25369-25375, 1993. Hekmetpanah DS and Young RA. Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection. Mol Cell Biol. 11: 57815791, 1991. Helmann JD and Chamberlin MJ. Structure and function of bacterial σ factors. Ann Rev Biochem. 57: 839-872, 1988. Honore NT, Bergh S, Chanteau S, Doucet-Populaire F, Eiglmeier K, Garnier T, Geroges C, Lanois P, Limpaipoon T, Newton S, Niang K, Portillo P, Ramesh GR, Reddi P, Ridel PR, Sittisombut N, Wu-Hunter S and Cole ST. Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: Structure and function of the Rif-Str regions. Mol Microbiol. 7: 207-214,1993. Hu JC and Gross CA. Marker rescue with plasmids bearing deletions in rpoD identifies a dispensible part of E. coli σ factor. Mol Gen Genet. 199: 7-13, 1983. Igarashi K, Fujita N and Ishihama A. Sequence analyses of two temperature-sensitive mutations in the α subunit gene (rpoB) of E. coli RNA polymerase. Nucl Acid Res. 18: 5945-5948, 1990. Igarashi K, Hanamura A, Makino K, Aiba H, Mizuno T, Nakata A and Ishihama A. Functional map of the α subunit of E. coli RNA polymerase: Two modes of transcription activation by positive factors. P roc Natl Acad Sci. USA 88: 8958-8962, 1991. Igarashi K and Ishihama A. Bipartite functional map of the E. coli RNA polymerase α subunit: Involvement of the C-terminal region in transcription activation by cAMPCRP. Cell. 65: 1015-1022, 1991. Ishihama A. Functional Modulation of Escherichia coli RNA polymerase. Annu Rev Microbiol. 54: 499-518, 2000.

Ishihama A, Shimamoto N, Aiba H, Kawakami K, Nashimoto H, Tsugawa A and Uchida N. Temperaturesensitive mutations in the α subunit gene of E. coli RNA polymerase. J Mol Biol. 137: 137-150, 1980. Ito K, Egawa K and Nakamura Y. Genetic interaction between the β' subunit of RNA polymerase and the arginine-rich domain of E. coli nusA protein. J Bacteriol. 173: 1492-1501, 1991. Iwabe N, Kuma KK, Kishino H, Hasegawa M and Miyata T. Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria. J Mol Evol. 32: 70-78, 1991. Jin DJ. Slippage synthesis at the galp2 promoter of E. coli and regulation by UTP concentration and cAMP-centerdot-cAMP protein. J Biol Chem. 269: 17221-17227, 1994. Jin DJ and Gross CA. RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation. J Biol Chem. 266: 11178-14485, 1991. Jin DJ and Gross CA. Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants. Mol Gen Genet. 216: 269275, 1989. Jin DJ and Gross CA. Mapping and sequencing of mutations in the E. coli rpoB gene led to rifampicin resistance. J Mol Biol. 202: 45-48, 1988. Jin R, Sharif KA and Krakow JS. Evidence for contact between the cyclic AMP receptor protein and the σ70 subunit of E. coli RNA polymerase. J Biol Chem. 270: 19231-19216, 1995. Jones CH and Moran CP. Mutant σ factor blocks transition between promoter binding and initiation of transcription. P roc Nat Acad Sci. USA 89: 1958-1962, 1992. Kashlev M, Lee J, Zalenskaya K, Nikivorov V and Goldfarb A. Blocking of the initiation to elongation transition by a transdominant RNA polymerase mutation. Science. 1006-1009, 1990 Kashlev M, Martin E, Polyakov A, Severinov K and Nikiforov A. Histidine-tagged RNA-polymerasedissection of the transcription cycle using immobilized enzyme. Gene. 130: 9-14, 1993. Kawakami K and Ishihama A. Defective assembly of ribonucleic acid polymerase subunits in a temperaturesensitive α subunit mutant of E. coli. Biochem. 19: 34913495, 1980. Khan D and Ditta G. Molecular structure of FixJ: Homology of the transcriptional activator domain with the –35 binding domain of σ factors. Mol Microbiol. 5: 987-997, 1991. Kimura M, Fujita N and Ishihama A. Functional map of the α subunit of E. coli RNA polymerase-deletion analyses of the amino-terminal assembly. J Mol Biol. 242: 107115, 1994. Kimura M and Ishihama A. Functional map of the α subunit

E. coli RNA polymerase of E. coli RNA polymerase-insertion analyses of the amino-terminal assembly. J Mol Biol. 248: 756-767, 1995. Kirkegaard K, Buc H, Spassky A and Wang JC. Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes. P roc Natl Acad Sci. USA 80: 2544-2548, 1983. Kumar A, Grimes B, Fujita N, Makino K, Malloch RA, Hayward RS and Ishihama A. Role of the σ70 subunit of E. coli RNA polymerase in transcription activation. J Mol Biol. 235: 405-413, 1994. Krummel B and Chamberlin MJ. Structural analysis of ternary complexes of E. coli RNA polymerase: deoxyribonuclease I footprinting of defined complexes. J Mol Biol. 225: 239-250, 1992. Landick R, Stewart J and Lee DN. Amino acid changes in conserved regions of the β subunit of E. coli RNA polymerase alter transcription pausing and termination. Genes Dev. 4: 1623-1636, 1990. Lee J and Goldfarb A. lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter. Cell. 66: 793-798, 1991. Lee HS, Ishihama A and Kustu S. The C-terminus of the asubunit of RNA-polymerase is not essential for transcriptional activation of σ54 holoenzyme. J Bacteriol. 175: 2479-2582, 1993. Lesley SA and Burgess RR. Characterisation of the E. coli transcription factor σ70: localisation of a region involved in the interaction with core RNA polymerase. Biochem. 28: 7728-7734, 1989. Lisitsyn NA, Sverdlov ED, Moiseyeva EP, Danilevskaya ON and Nikiforov VG. Mutation to rifampicin resistance at the beginning of the RNA polymerase β subunit gene in E. coli. Mol Gen Genet. 196: 173-174, 1984. Lonetto M, Gribskov M and Gross C. The σ70 family: Sequence conservation and evolutionary relationships. J Bacteriol. 174: 3843-3849, 1992. Lou J and Krakow JS. Characterization and epitope mapping of monoclonal antibodies directed against the β' subunit of Escherichia coli RNA polymerase. J Mol Chem. 267: 18175-18181, 1992. Makino K, Amemura M, Kim SK, Nakata A and Shinagawa H. Role of the σ70 subunit of E. coli RNA polymerase in transcription activation by activator protein phoB in E. coli. Genes Dev. 7: 149-160, 1993. Malik T, Ahmad K, Büyükuslu N, Cromie K, and Glass RE. Intragenic suppression of trans-dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C-terminus. Genes to Cells. 4: 501-515, 1999. Merrick M and Chambers S. The helix-turn-helix motif of σ54 is involved in recognition of the –13 promoter element. J Bacterial. 174: 7221-7226, 1992.

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Merrick M, Gibbins J and Toukdarian A. The nucleotide sequence of the σ factor gene ntr (rpoN) of Azobacter vinelandii: analysis of conserved sequences in NtrA proteins. Mol Gen Genet. 210: 323-330, 1987. Miller LP, Crawford JT and Shinnick TM. The rpoB gene of mycobacterium-tuberculosis. Antimicrobial Agents and Chemotherapy. 38: 805-811, 1994. Mukherjee K and Chatterji D. Alteration in template recognition by Escherichia coli RNA polymerase lacking the ω subunit: A mechanistic analysis through gel retardation and foot-printing studies. J Bio Sci. 24: 453-461, 1999 Mukherjee K, Nagai H, Shimamoto N and Chatterji D. GroEL is involved in activation of E. coli RNA polymerase devoid of σ subunit in vivo. Eur J Biochem. 266: 228-235, 1999. Murakami KS, Masuda S and Darst S. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4Å resolution. Science. 296(5571): 1280-1284, 2002. Mustaev A, Kashlev M, Polyakov J, Lebedev A, Zalenskataya K, Grachev M, Goldfarb A and Nikiforov V. Mapping of the priming substrate contacts in the active-centre of E. coli RNA polymerase. J Biol Chem. 266: 23927-23931, 1991. Mustaev A, Kashlev M, Zaychikov E, Grachev M, and Goldfarb A. Active-center rearrangement in RNA polymerase initiation. J Biol Chem. 268: 19185-19187, 1993. Mustaev A, Zaychikov E, Severinov K, Kashlev M, Polyakov A, Nikiforov V and Goldfarb A. Topology of the RNA-polymerase active center probed by chimeric rifampicin-nucleotide compounds. P roc Natl Acad Sci. USA 91: 12036-12040, 1994. Naryshkina T, Mustaev A, Darst SA and Severinov K. The β' subunit of E. coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin. J Biol Chem. 276: 1330813313, 2001. Nene V and Glass RE. Genetic studies on the β subunit of E. coli RNA polymerase IV Structure-function correlates. Mol Gen Genet. 194: 166-172, 1984. Ovchinnikov YA, Monastyrskaya GS, Guriev VV, Chertov OY, Modyanov NN, Grinkevich VA, Makarova IA, Marchenko TV, Polovnikova IN, Lipkin VM and Sverdlov ED. The primary structure of E. coli RNA polymerase nucleotide-sequence of the rpoB gene and amino acid sequence of the subunit. Eur J Biochem. 116: 621-629, 1981. Ovchinnikov YA, Monastyrskaya GS, Guriev SO, Kalinina NF, Sverdlov ED, Gragerov AI, Bass IA, Kiver IF, Moiseyeva EP, Igumnov VN, Mindlin SZ, Nikiforov VG and Khesin RB. RNA polymerase rifampicin resistance mutations in E. coli: Sequence changes and dominance. Mol Gen Genet. 190: 344-348, 1983.

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Ovchinnikov YA, Monastryskaya GS, Gubanov VV, Guryev SO, Salomanita IS, Shuvaeva TM, Lipkin VM and Sverdlov ED. The primary structure of E. coli RNA polymerase. Nucleotide sequence of the rpoC gene and amino acid sequence of the b' subunit. Nucl Acid Res. 10: 4035-4044, 1982. Panny SR, Heil A, Mazus B, Palm P, Zillig W, Mindlin SZ. Ilyina TS and Khesin RB. A temperature sensitive mutation of the β' subunit of DNA-dependent RNA polymerase from E. coli T116. FEBS letter. 48: 241-245, 1974. Petersen SK and Flemming GH. A missense mutation in the rpoC gene affects chromosomal replication control in E. coli. J Bacteriol. 173: 5200-5206, 1991. Polyakov A, Nikiforov V and Goldfarb A. Disruption of substrate binding site in E. coli RNA polymerase by lethal alanine substitutions in carboxy terminal domain of the β subunit. FEBS letter. 444: 189-194, 1999. Polyakov A, Severinova E and Darst SA. 3-dimensional structure of E. coli core RNA-polymerase-promoter binding and elongation conformations of the enzyme. Cell. 83: 365-373, 1995. Popham DL, Szeto D, Keener J and Kustu S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 243: 629-635, 1989. Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K and Gourse RL. A third recognition element in bacterial promoters-DNAbinding by α subunit of RNA polymerase. Science. 262: 1407-1413, 1993. Sasse-Dwight σ and Gralla JD. Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor σ54. Cell 62: 945-954, 1990. Severinov K, Fenyo D, Severinova E, Mustaev A, Chait BT and Goldfarb DS. The σ-subunit conserved region-3 is part of 5'-face of active-center of E. coli RNApolymerase. J Biol Chem. 269: 20826-20828, 1994. Severinov K, Mustaev A, Kashlev M, Borukhov S, Nikiforov V and Goldfarb A. Dissection of the β subunit in the E. coli RNA polymerase into domains by proteolytic cleavage. J Biol Chem. 267: 12813-12819, 1992. Severinov K, Mustaev A, Severinov E, Kozlov M and Darst SA. The β subunit rif-cluster-I is only angstroms away from the active center of E. coli RNA-polymerase. J Biol Chem. 270: 29428-29432, 1995. Severinov K, Soushko M, Goldfarb A and Nikiforov V. Rifampicin region revisited- new rifampicin-resistant and streptolydigin-resistant mutants in the β subunit of E. coli RNA polymerase. J Biol Chem. 268: 1482014825, 1993. Siebenlist U, Simpson R and Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 20: 269-272, 1980.

Siegele DA, Hu JC, Walter WA and Gross CA. Altered promoter recognition by mutant forms of the σ70 subunit of E. coli RNA polymerase. J Mol Biol. 206: 591-603, 1989. Sparkovski J and Das A. Simultaneous gain and loss of functions caused by a single amino acid substitution in the β subunit of E. coli RNA polymerase: Suppression of NusA and rho mutations and conditional lethality. Genetics. 130: 411-428, 1992. Straiger S, Kunkel B, Kroos L, and Losick R. Chromosomal rearrangement generating a composite gene for a developmental σ factor. Science. 243: 507-512, 1989. Straiger P, Parsot C, and Bouvier J. Two functional domains conserved in major and alternate bacterial σ factors. FEBS letters. 187: 11-15, 1985. Squires CH, Defelice M, Wessler SR and Calvo JM. Physical characterisation of the ilvH1 operon of E. coli K12. J Bacteriol. 147: 797-804, 1981. Sweetser D, Nonet M and Young RA. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. P roc Natl Acad Sci. USA 84: 1192-1196, 1987. Tatti KM, Jones CH and Moran PC. Genetic evidence for interaction of σE with the spoIIID promoter in B. subtilis. J Bacteriol. 173: 7828-7833, 1991. Thomas M and Glass RE. E. coli rpoA mutation which impairs transcription of positively regulated systems. Mol Microbiol. 5: 2719-2725, 1991. Tichelar W and Heel MV. Characteristic views of E. coli RNA polymerase core enzyme in the scanning transmission electron microscope. J Structural Biol. 103: 180-184, 1990. Toketo M and Ishihama A. Biosynthesis of RNA polymerase in E. coli IV. Accumulation of intermediates in mutants defective in the subunit assembly. J Mol Biol. 102: 297310, 1976. Vassylyev DG, Sekine S, Laptenko O, Lee J, Vassylyeva MN, Borukhov σ and Yokoyama S. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6Å resolution. Nature. 417: 712-719, 2002. Waldburger C, Gardella T, Wang R and Suskind MM. Changes in conserved region 2 of E. coli σ70 affecting promoter recognition. J Mol Biol. 215: 267-276, 1990. Weilbaecher R, Hebron C, Feng GH and Landick R. Termination-altering amino-acid substitutions in the β' subunit of Escherichia coli RNA-polymerase identify regions involved in RNA chain elongation. Genes Dev. 8: 2913-2927, 1994. Wellman A and Meares CF. Footprint on the sigma protein: a re-examination. Biochem Biophys Res Comm. 177: 140-144, 1991. Wigneshweraraj SR, Nechaev S, Severinov K and Buck M. The β subunit residues 186-433 and 436-445 are commonly used by σ54 and σ70 RNA polymerase holoenzyme for open promoter complex formation. J Mol Biol. 319: 1067-1083, 2002.

E. coli RNA polymerase Zakharova N, Bass A, Arsenieva E, Nikiforov V and Severinov K. (submitted for publication). Mutations in and monoclonal antibody binding to evolutionary hypervariable region of E. coli RNA polymerase β' subunit inhibit transcript cleavage and transcript elongation. J Biol Chem. 2003. Zillig W, Zechel K, Rabussay D, Schachner M, Sethi VS, Palm P, Heil A and Seifert W. On the role of different subunits of DNA-dependenet RNA polymerase from E. coli in the transcription process. Cold Spring Harbor Symp Quant Biol. 35: 47-58, 1970. Zuber P, Healy J, Carter HL, Cutting S, Moran CP and Losick R. Mutation changing the specificity of an RNA polymerase σ factor. J Mol Biol. 206: 605-614, 1989.

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