A Coiled-Coil from the RNA Polymerase β′ Subunit Allosterically Induces Selective Nontemplate Strand Binding by σ70 (original) (raw)

eral transcription factors (e.g., TFIIB, TFIID, TFIIF, etc. for RNA polymerase II) facilitate initiation in eukaryotes, whereas a single protein, called , serves as an initiation factor in prokaryotes (Orphanides et al., 1996; Record et al., 1996). Several alternative sigma factors are presmediated by 70 , the primary or housekeeping sigma University of California, San Francisco factor in Escherichia coli (Gross et al., 1992). San Francisco, California 94143 70 holoenzyme recognizes promoters characterized 2 Department of Bacteriology and Department by two conserved hexamers centered at Ϫ10 and Ϫ35 of Oncology relative to the start site of transcription, and then melts University of Wisconsin, Madison promoters roughly between Ϫ11 to ϩ1, a process called Madison, Wisconsin 53706 open complex formation. Though the structural mecha-3 Departments of Stomatology and Microbiology nism of this process is unknown, several discrete activiand Immunology ties have been defined: binding to double-stranded DNA University of California, San Francisco in the promoter (Gross et al., 1998), binding to the junc-San Francisco, California 94143 tion between double-stranded and single-stranded DNA, 4 E.A. Doisy Department of Biochemistry (Guo and Gralla 1998; Matlock and Heyduk, 2000), and and Molecular Biology binding to the nontemplate strand of the Ϫ10 region of St. . Although the initiation factor 70 provides most or all of the specific recognition determinants Summary for each of these processes, free 70 is unable to perform any of these activities by itself (Kenney et al., 1989; For transcription to initiate, RNA polymerase must rec-Siegele et al. 1989; Daniels et al., 1990; Waldburger et ognize and melt promoters. Selective binding to the al., 1990; Dombroski et al., 1992, 1993, 1997; Severinova nontemplate strand of the Ϫ10 region of the promoter et al., 1996; Marr and Roberts, 1997; Naryshkin et al., is central to this process. We show that a 48 amino 2000). Rather, core RNA polymerase (core) is required acid (aa) coiled-coil from the ␤ subunit (aa 262-309) and is likely to contribute at least two functions: uninduces 70 to perform this function almost as effimasking recognition determinants in sigma and strengthciently as core RNA polymerase itself. We provide eviening binding by providing additional nonspecific DNA dence that interaction between the ␤ coiled-coil and interactions. We would like to dissect the very large (450 region 2.2 of 70 promotes an allosteric transition that kDa) holoenzyme into the minimal portions capable of allows 70 to selectively recognize the nontemplate reconstituting each activity to facilitate future biochemistrand. As the ␤ 262-309 peptide can function with cal and structural study of open complex formation. the previously crystallized portion of 70 , nontemplate Selective binding of holoenzyme to the nontemplate recognition can be reconstituted with only 47 kDa, or strand of the Ϫ10 region of the promoter is likely to 1/10 of holoenzyme. capture, stabilize, and extend transient strand separation. We therefore focused our attention on determining Introduction the minimal assembly of 70 and core that could carry out this activity. There is good understanding of the Transcription of RNA is a crucial part of gene expression regions of 70 required for this process. The 70 family in all organisms. Transcription initiation requires that of proteins has four regions of sequence conservation RNA polymerase recognize and then locally unwind prowhich have been divided into subregions (see Figure 5A moter DNA to gain access to the template strand. This for a description of these conserved regions; Lonetto process is carried out as a collaboration between RNA et al., 1992). When bound to core, the fragment of 70 polymerase and its initiation factors, a form of RNA polywhich has been crystallized (which extends from the merase called holoenzyme. The multisubunit RNA polymiddle of conserved region 1 to the end of conserved merase is conserved throughout evolution (Young, 1991; region 2.4) specifically recognizes the nontemplate Sentenac et al., 1992; Ebright, 2000). The five subunits strand (Severinova et al., 1996; Malhotra et al. 1996). of prokaryotic RNA polymerase (␤Ј,␤, ␣ 2 , and ) are all Within this fragment, region 2.4 has at least some of the conserved in eukaryotic polymerases, though the latter recognition determinants for nontemplate strand bindcontain several additional subunits (Young 1991; Sening as mutational alterations in 2.4 that broaden protenac et al., 1992; Ebright, 2000; Minakhin et al., 2001). moter specificity also broaden recognition of the non-In contrast, initiation factors are distinct in prokaryotic template strand (Marr and Roberts, 1997). From the core and eukaryotic organisms. A set of proteins called genside, it appears that the isolated ␤Ј subunit, together with 70 , is sufficient for selective recognition of nontemplate strand (Kulbachinskiy et al., 1999). NIH (GM28575) to R.R.B. B.A.Y. was supported by NSF predoctoral Ko, D.C., Marr, M.T., Guo, J., and Roberts, J.W. (1998). A surface of Escherichia coli sigma 70 required for promoter function and fellowship DGE-9616282.