Proteopedia entry: Beta-prime subunit of bacterial RNA polymerase (original) (raw)
Related papers
Cell, 2002
to this multistep process: bacterial RNAP core enzyme (Zhang et al., 1999; subunit composition Ј//␣ I /␣ II /), eukaryotic RNAP II core enzyme ⌬4/7 (Cramer et al., 2001; subunit composition 1/2/3/5/6/8/9/10/11/12, where 1, 2, 3, 11, and 6 are homologs of bacterial RNAP and ; Ebright, 2000), and a eukaryotic RNAP II tailed-template elongation complex .
The Structure of Bacterial RNA Polymerase
ASM Press eBooks, 2014
In this review, we will describe recent advances in RNAP structure and their implications for understanding the mechanism of transcription and the regulation of key steps in the transcription cycle. To lay a foundation for understanding the structures, we begin with a summary of the main features of the transcription cycle, RNAP's mechanism, and RNAP's subunit composition and primary structure. The transcription cycle There are three main steps in the transcription cycle: initiation, elongation and termination. During initiation, the core RNAP enzyme (subunit composition 2 , , and in bacteria; reference (13) binds to one of the family of initiation factors. The resulting holoenzyme is able to bind specifically to the promoter DNA, forming the closed complex (RP c ; references 14, 47) in a process called promoter recognition (Fig. 1A). RP c isomerizes in two or more steps to the open complex (RP o), in which the strands of the DNA have melted to allow active-site access to the template strand (78). RP o is capable of initiating transcription, but, in most cases, remains at the promoter in an initial transcription complex (ITC) that undergoes reiterative rounds of short transcript formation and release, called abortive transcription (Fig. 1A), before releasing contacts with the DNA and escaping from the promoter (48, 98). After RNAP leaves the promoter, it forms a transcription elongation complex (TEC), the subunit is bound less avidly, and eventually dissociates. The TEC is processive and extremely stable (49), transcribing at an average rate of 30-100 nt/sec for tens of kilobases down the DNA template (54, 99). Transcription ends when RNAP reaches an
Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments
Proceedings of the National Academy of Sciences, 1995
The Escherichia coli rpoB gene, which codes for the 1342-residue j3 subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP 1 subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these p-subunit polypeptide fragments, when added with purified recombinant P', or, and a subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the 1f subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.
Structural basis for substrate loading in bacterial RNA polymerase
2007
The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-Å resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-59-[(a,b)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active ('insertion') site closed through refolding of the trigger loop (TL) into two a-helices. In contrast, the EC/AMPcPP/ streptolydigin structure reveals an inactive ('preinsertion') substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.
RNA polymerase: the vehicle of transcription
Trends in Microbiology, 2008
RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation for all three divisions of life: Eukaryota, Archaea and Bacteria. Recent progress in the structural and biochemical characterization of RNAP illuminates this enzyme as a flexible, multifunctional molecular machine. During each step of the transcription cycle, RNAP undergoes elaborate conformational changes. As many fundamental and previously mysterious aspects of how RNAP works begin to be understood, this enzyme reveals intriguing similarities to man-made engineered devices. These resemblances can be found in the mechanics of RNAP-DNA complex formation, in RNA chain initiation and in the elongation processes. Here we highlight recent advances in understanding RNAP function and regulation. Review Glossary cAMP: cyclic adenosine monophosphate. CAP: catabolite activator protein. F(bridge)-helix and G(trigger)-loop: mobile elements of the RNAP catalytic center (sometimes also called 'bridge helix' and 'trigger loop', respectively). i+1: the substrate binding site in the RNAP catalytic center. P lac : promoter of the lactose operon. ppGpp: guanosine tetraphosphate, the effector of the stringent response. RNAP: RNA polymerase. RPc: RNAP-promoter closed complex. RPitc: RNAP-promoter initial transcribing complex. RPo: RNAP-promoter open complex. aCTD: carboxy-terminal domain of alpha subunit. s1, s2, s3, s4: evolutionarily conserved domains of s 70. s3-s4 linker: sigma subunit linker element connecting domains s3 and s4. s 54 : sigma N, the sigma factor regulating nitrogen metabolism. s 70 : sigma 70, the housekeeping sigma factor in Escherichia coli.
Electron Crystal Structure of an RNA Polymerase II Transcription Elongation Complex
Cell, 1999
nel in yeast RNA polymerase II was suggested to retain DNA and thereby enhance the processivity of a tran-Stanford University School of Medicine Stanford, California 94305 scribing complex (Darst et al., 1991a). Consistent with this idea, electron crystallography revealed two alternative conformations of the arm in E. coli RNA polymerase: an open conformation allowing access to the channel Summary in the polymerase holoenzyme (Darst et al., 1989), the form responsible for initiation; and a closed conforma-The structure of an actively transcribing complex, contion, with the arm around the channel, in core polymertaining yeast RNA polymerase II with associated temase (Polyakov et al., 1995), the form of the enzyme inplate DNA and product RNA, was determined by elecvolved in RNA chain elongation. The occurrence of an tron crystallography. Nucleic acid, in all likelihood the open form of yeast RNA polymerase II under conditions "transcription bubble" at the active center of the enconducive to initiation has been demonstrated as well zyme, occupies a previously noted 25 Å channel in the (Asturias et al., 1997; Asturias and Kornberg, 1999). The protein structure. Details are indicative of a roughly 90؇ two conformations of the yeast and E. coli RNA polymerbend of the DNA between upstream and downstream ases have so far been seen, however, only in the abregions. The DNA apparently lies entirely on one face sence of DNA. The proposal that a transcribing polymerof the polymerase, rather than passing through a hole ase adopts the closed conformation remains to be to the opposite side, as previously suggested. tested. Additional features of yeast RNA polymerase II have been identified by difference electron crystallography. Two small subunits, Rpb4 and Rpb7, were located in a Introduction niche in the floor of a 25 Å groove, prompting speculation as to their role in polymerase-DNA interaction (Jensen RNA polymerase II, responsible for all mRNA synthesis et al., 1997). Binding sites for two transcription initiation in eukaryotes, is a complex of over half a million daltons factors, TFIIB and TFIIE, were identified at a distance composed of 12 different subunits, conserved across from the 25 Å channel and in a position relative to the species from yeast to humans (Young, 1991). The two arm around the channel with important implications for largest subunits have counterparts in the four-subunit DNA binding (Leuther et al., 1996). The question of bacterial RNA polymerase as well. Despite the size and whether DNA does occupy the channel during transcripcomplexity of RNA polymerase II, additional protein faction is crucial for understanding the roles of these and tors are required for initiation and for aspects of RNA other accessory factors. chain elongation. This constellation of transcription pro-DNase I protection mapping of RNA polymerase II teins presents a formidable challenge for structural transcription elongation complexes has shown the asanalysis. sociation of 40-50 base pairs of DNA with the enzyme, Structures of yeast RNA polymerase II and of E. coli roughly centered on the active site (Rice et al., 1993; RNA polymerase have been determined from two-Selby et al., 1997). This length of DNA, sufficient to wrap dimensional (2D) crystals by electron microscopy and nearly all the way around the polymerase, far exceeds image processing at 16-24 Å resolution (Darst et al., the size of the 25 Å channel. There is also evidence 1989, 1991a; Schultz et al., 1993; Polyakov et al., 1995; from nuclease protection and direct binding studies for Jensen et al., 1997). A notable feature of the enzymes interaction of polymerase II with about 20 residues of at this resolution is a 25 Å channel, appropriate in size RNA (Rice et al., 1991; Johnson and Chamberlin, 1994; for binding duplex DNA. A similar feature in the X-ray Gu et al., 1996). The problem of polymerase-nucleic acid structures of single-subunit DNA and RNA polymerases interaction therefore goes well beyond the question of harbors the active center of the enzymes. Biochemical whether DNA binds in the channel. studies have indicated, however, that the active center of the multisubunit RNA polymerases is not closely re-We report here on difference electron crystallography lated to those of the single-subunit enzymes (Treich et between RNA polymerase II-nucleic acid complexes al., 1992; Markovtsov et al., 1996; Mustaev et al., 1997), and the polymerase alone. Initial studies, performed with polymerase-DNA template complexes, were hampered by a low yield and poor quality of 2D crystals, attributed * To whom correspondence should be addressed (e-mail: kornberg@ to interference by free DNA. The work was therefore stanford.edu). extended to paused elongation complexes in which the † These authors contributed equally to this work. nucleic acids are more tightly bound. The results shed ‡ Present address: Caliper Technologies Corp., 605 Fairchild Drive, light on a number of issues, including the role of the Mountain View, California 94043-2234.
Journal of Biological Chemistry, 1995
Escherichia coli DNA polymerase III holoenzyme in the presence of ATP and E. coli single-stranded DNAbinding protein forms an initiation complex on a primed template capable of rapid and highly processive DNA replication. DNase I digestion of initiation complexes demonstrated that holoenzyme protected 27-30 nucleotides of primer. Like the formation of initiation complexes, this protection required both ATP and E. coli single-stranded DNA-binding protein.