Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases (original) (raw)
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Wrapping of DNA around the E.coli RNA polymerase open promoter complex
The EMBO Journal, 1999
High-resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase·σ 70 (RNAP) open promoter complex (RP o ). A detailed analysis of a large number of molecules shows that the DNA contour length of RP o is reduced by~30 nm (~90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88°. The contour length reduction and the DNA bend angle were much less in inactive RNAP-DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300°around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two-thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one-third is part of the downstream region. Based on these data, a model of the σ 70 ·RP o conformation is proposed. Keywords: atomic force microscopy (AFM)/DNA bending/DNA wrapping/open promoter complex/RNA polymerase/transcription
Study of elongation complexes for T7 RNA polymerase
Biophysics, 2012
Complexes of bacteriophage T7 RNA polymerase with a DNA template for transcription elonga tion were visualized by atomic force microscopy. Images for complexes of T7 RNA polymerase with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template mole cule with several T7 RNA polymerase molecules corresponding to stages of initiation, elongation and termi nation of transcription were visualized under the elimination of unspecific DNA protein binding. Immobi lized on the amino mica RNA transcripts form rod like condensed structures. Detailes of specific and unspe cific complex formation for the T7 RNA polymerase-DNA system during initiation and transcription elongation are discussed.
Biochemistry, 2000
The subunits of Saccharomyces cereVisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3′-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N 3 RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N 3 RdUTP, which has a photoreactive azido group 9 Å from the uridine base, we used the photoaffinity cross-linker 5N 3 dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N 3 RdUTP. With 5N 3 dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex. triphosphate; RNAP, RNA polymerase; RPB1-RPB12, yeast RNA polymerase II subunits; RPC, yeast RNA polymerase III subunit; TE, 10 mM Tris-HCl (pH 7.9) and 0.1 mM EDTA.
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.
Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex
Nucleic Acids Research, 1993
Elongation complexes of RNA polymerase II, RNA-DNAenzyme ternary complexes, are Intermediates In the synthesis of all eukaryotlc mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and Its Interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes Is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyrlbonuclease I (DNAse I) to map the Interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzymetemplate Interactions of Individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformatlonal transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.
Engineering of Elongation Complexes of Bacterial and Yeast RNA Polymerases
Methods in Enzymology, 2003
Certain DNA sequences induce pausing, arrest, or termination of transcription 1 modulating catalytic activity and stability of the elongation complex (EC) between RNA polymerase (RNAP), template DNA, and nascent RNA. 2 The ECs of bacterial RNAP and eukaryotic RNAP II (Pol II) have similar structure, in which the enzyme covers 30-35 nucleotides (nt) of the double-stranded DNA containing $12-nt melted segment called the transcription bubble. 2,3 Between 8-9 nt of the 3 0 -proximal RNA hybridize with the template DNA strand within the bubble. 4,5 The transcript exits RNAP at a distance of 14 nt from its 3 0 end; 6 therefore, 5-6 nt of single-stranded RNA upstream from the hybrid are located inside the enzyme. 7 Although RNAP forms multiple contacts with RNA and DNA within the protected regions, only RNA:DNA hybrid and 9-12 nt of the DNA duplex downstream from the RNA 3 0 end are needed for high stability of bacterial EC. 8 Surprisingly, the downstream DNA duplex is dispensable for stability of the EC formed by yeast Pol II. 9 Elongation of RNA is accompanied by stepwise forward translocation of RNAP along the template. In addition, RNAP is capable of backward movement, which is induced by degradation of the transcript from the 3 0 end, either by pyrophosphorolysis 10 (a reaction reverse to nt addition) or by endonucleolytic cleavage stimulated by protein factors GreA and GreB. 11 Also, at certain DNA sequences RNAP moves backward along the RNA and DNA without any shortening of the transcript. 12 This translocation, or 1 R.
European Journal of Biochemistry, 1986
DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in nondenaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between CJ and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5: 1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and CJ factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the CJ factor.
The EMBO Journal, 2002
contributed equally to this work Analysis of multisubunit RNA polymerase (RNAP) structures revealed several elements that may constitute the enzyme's functional sites. One such element, the`rudder', is formed by an evolutionarily conserved segment of the largest subunit of RNAP and contacts the nascent RNA at the upstream edge of the RNA±DNA hybrid, where the DNA template strand separates from the RNA transcript and re-anneals with the non-template strand. Thus, the rudder could (i) maintain the correct length of the RNA±DNA hybrid; (ii) stabilize the nascent RNA in the complex; and (iii) promote or maintain localized DNA melting at the upstream edge of the bubble. We generated a recombinant RNAP mutant that lacked the rudder and studied its properties in vitro. Our results demonstrate that the rudder is not required for establishment of the upstream boundary of the transcription bubble during promoter complex formation, nor is it required for separation of the nascent RNA from the DNA template strand or transcription termination. Our results suggest that the rudder makes critical contributions to elongation complex stability through direct interactions with the nascent RNA.
Conformational Heterogeneity in RNA Polymerase Observed by Single-Pair FRET Microscopy
Biophysical Journal, 2006
Kinetic, structural, and single-molecule transcription measurements suggest that RNA polymerase can adopt many different conformations during elongation. We have measured the geometry of the DNA and RNA in ternary elongation complexes using single-pair fluorescence resonance energy transfer. Six different synthetic transcription elongation complexes were constructed from DNA containing an artificial transcription bubble, an RNA primer, and core RNA polymerase from Escherichia coli. Two different RNA primers were used, an 8-mer and a 59-extended 11-mer. Fluorescent dye labels were attached at one of three positions on the DNA and at the RNA primer 59-end. Structurally, the upstream DNA runs perpendicular to the proposed RNA exit channel. Upon nucleoside-triphosphate addition, DNA/RNA hybrid separation occurs readily in the 11-mer complexes but not in the 8-mer complexes. Clear evidence was obtained that RNA polymerase exists in multiple conformations among which it fluctuates.