Spatial Organization of Transcription Elongation Complex in
            <i>Escherichia coli</i>

We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA–DNA, DNA–RNA, and RNA–RNA base pairing associated with ( i ) the translocational and size fluctuations of the transcription bubble; ( ii ) changes in the associated DNA–RNA hybrid; and ( iii ) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process.

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“…The basis for our quantitative analysis is the structural and mechanistic model of the elongation complex (EC) (3,5,10,23), sketched in Fig. 1.…”

Section: Model Of Transcription Elongationmentioning

confidence: 99%

Thermodynamic and kinetic modeling of transcriptional pausing

Tadigotla

Maoiléidigh

Sengupta

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…In the RNA exit channel, the interactions between nascent RNA and pol II in the region between 10 and 17 nt are expected to be very strong (30), for it is known that such interactions contribute to the stability of an elongation complex in prokaryotic RNA polymerase. Recent structural studies on the complexes formed by TFIIB with pol II have shown that the N-terminal segment of TFIIB, termed the ''B finger,'' reaches into the catalytic center of pol II exactly through a groove (31)(32)(33)(34), once presumed to be the RNA exit channel and proved to be true in this study.…”

Section: Discussionmentioning

confidence: 99%

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Chen¹,

Chang²,

Yen³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5 end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5 end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.nanometry ͉ structure ͉ transcription ͉ in-gel ͉ single-molecule fluorescence R NA polymerase II (pol II), a protein complex containing 12 subunits, Rpb1-Rpb12, of a total mass of Ϸ500 kDa and size Ϸ100-140 Å, is the enzyme machinery synthesizing mRNA in all eukaryotes (1). X-ray studies of pol II complexes (2-4) led to an atomic model containing structural elements with functional implications (Fig. 1A). In a transcribing pol II, between the ''clamp'' and ''jaw'' domain, lies a cleft (4) that harbors the active center, a straight duplex DNA and an RNA-DNA hybrid (position ϩ1 to Ϫ8, ϩ1 denoting the nucleotide addition site). The strand separation of RNA from DNA template occurs upstream of the hybrid at positions Ϫ9 and Ϫ10, facilitated by a set of protein loops including the ''lid'' domain as a driving wedge. Nascent RNA moves through an exit pore from the active center, crossing a saddle-like surface, beneath an ''arch'' bridging the clamp and wall (5).How does pol II instruct the nascent RNA to exit beyond the saddle? Is there a unique path on pol II connecting the active center to its exterior that nascent RNA may follow? To date, insights into the RNA exit have come from analysis of pol II surface charge distribution: two positively charged grooves, on either side of the ''dock domain'' (Fig. 1 A), can accommodate ssRNA (5). One groove, putatively referred as ''exit channel 1,'' runs around the base of the clamp, leading toward the stalk of subcomplex Rpb4-Rpb7, which can bind RNA via its ribonucleoprotein fold (6, 7). The other groove, termed ''exit channel 2,'' runs down the back side of pol II, through Rpb3 and Rpb11, leading toward Rpb8, a subunit equally competent in RNA binding by its single-strand nucleic acid-binding motif. Intriguingly, exit channel 1 would cause the RNA to bend sharply, implying that channel 2 is energetically favored for RNA binding. Yet, evidence in support of the channel 1 hypothesis has come from observation...

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“…Macromolecular interactions among DNA, RNA, and E. coli RNA polymerase in the elongation complexes have been mapped (2)(3)(4)(5). A double clamp model has been proposed for elongating E. coli RNA polymerase where DNA entry and RNA exit sites are directed opposite to each other in the same region (5).…”

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confidence: 99%

Two Site Contact of Elongating Transcripts to Phage T7 RNA Polymerase at C-terminal Regions

Shen

Kang

2001

Journal of Biological Chemistry

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A series of active elongation complexes of the phage T7 RNA polymerase were obtained through stepwise walking of the polymerase along an immobilized DNA template. Transcripts were radiolabeled at the 16th to 18th residues, and a photocross-linkable 4-thio-UMP was separately incorporated at the 22nd, 24th, 32nd, and 38th residues. Such complexes (up to 51 nucleotides) produced by the incorporation of one nucleotide at a time were isolated and individually subjected to long wave UV cross-linking. Only when the cross-linker was positioned at the 3-end (؊1) of the elongating RNA and 8 nucleotides upstream (؊9), was the RNA substantially cross-linked to the polymerase, regardless of how far it was from the 5-end of the transcripts. Linkage of the 3-end residue was mapped to the Thr 636 -Met 666 region, which contains nucleotide-binding sites. The ؊9 residue was cross-linked to the Ala 724 -Met 750 region rather than to the N-terminal region. These two contacts were maintained throughout the elongation complexes and reveal a route of nascent RNA through the T7 RNA polymerase in elongation complexes.The structural organization of transcription elongation complexes has been well characterized with histidine-tagged Escherichia coli RNA polymerase arrested at specific positions on templates (1). Macromolecular interactions among DNA, RNA, and E. coli RNA polymerase in the elongation complexes have been mapped (2-5). A double clamp model has been proposed for elongating E. coli RNA polymerase where DNA entry and RNA exit sites are directed opposite to each other in the same region (5). Experimental evidence (6, 7) also is consistent with this mechanism for RNA polymerase action.The T7 RNA polymerase, a single-subunit protein of 99 kDa, is capable of initiating transcription promoter-specifically, elongating with high processivity and terminating in the absence of additional factors. The mechanism of initiation and termination of T7 RNA polymerase has been studied extensively because of its simplicity of structure and reaction system. On the other hand, elongation has been little studied, mainly because a series of stable and active ternary elongation complexes have not been obtained with the T7 RNA polymerase.The overall three-dimensional structures of T7 RNA polymerase complexed with an open promoter (8) and a transcribing initiation complex (9) are similar to that of free enzyme (10).Some regions, including the active site, change into a more compact structure in the initiation complex. Structural information on elongation complexes with a growing transcript has not yet been determined.Several lines of evidence have suggested that the structure of the T7 RNA polymerase ternary complex changes when it proceeds from the initiation phase to the elongation phase. Both protease sensitivity (11) and the protected region of the DNA template strand in footprinting with methidiumpropyl-EDTA-Fe(II) greatly change (12, 13) during the switch from initiation to elongation. The length of the DNA-RNA hybrid also changes from 2-4 bp 1 i...

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Spatial Organization of Transcription Elongation Complex in Escherichia coli

Cited by 78 publications

References 15 publications

Thermodynamic and kinetic modeling of transcriptional pausing

Thermodynamic and kinetic modeling of transcriptional pausing

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Two Site Contact of Elongating Transcripts to Phage T7 RNA Polymerase at C-terminal Regions

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