The structural basis of the transition from initiation to elongation phases of transcription, as well as translocation and strand separation, by T7 RNA polymerase

doi:10.1016/j.sbi.2004.01.006

Current Opinion in Structural Biology

Volume 14, Issue 1, February 2004, Pages 4-9

https://doi.org/10.1016/j.sbi.2004.01.006 Get rights and content

Abstract

The RNA polymerase from phage T7 is a 99 kDa single polypeptide that is unrelated to the multisubunit cellular RNA polymerases, but exhibits nearly all of their properties. Six separate crystal structures have enhanced our understanding of promoter DNA recognition, duplex DNA opening, and the transition from the abortive initiation phase to the elongation phase. A major conformational change in the N-terminal domain removes the promoter-binding site, accounting for promoter clearance, and creates a tunnel through which the transcript passes, accounting for the processivity of the elongation phase. Structures of substrate and product complexes show that a rotational conformational change of the fingers domain is associated with translocation and downstream strand separation. The rotation that results in translocation is powered by the release of the pyrophosphate product.

Introduction

The 99 kDa single-subunit RNA polymerase from bacteriophage T7 (T7 RNAP) and the multisubunit cellular RNAPs share numerous functional characteristics, in spite of their being structurally unrelated. Both families of RNAPs have initiation and elongation phases of transcription, and must translocate down the DNA as synthesis proceeds, opening the downstream duplex DNA 1., 2.. In the initiation phase, RNAP binds to a specific DNA promoter sequence, opens the duplex at the transcription start site to form a bubble and initiates RNA synthesis de novo. Transcription during this phase is characterized by repeated abortive initiation attempts that produce short RNA fragments 2–6 nucleotides in length 3., 4.. After synthesis of a 10–12-nucleotide RNA, the polymerase enters the elongation phase and completes the RNA transcript processively without dissociation until termination. High-resolution crystal structures of T7 RNAP trapped in a transcribing initiation complex (Figure 1a) with DNA and a 3-nucleotide transcript [5], and an elongation complex (Figure 1b) containing 30 base pairs of DNA and 17 nucleotides of RNA [6^••], as well as a complex with downstream duplex and an 8-nucleotide RNA transcript [7^••], provide the structural basis of the opening of the promoter, the transition from abortive initiation to processive elongation phases, promoter clearance and the unwinding of downstream DNA.

RNA synthesis occurs through cycles of repeated ribonucleotide incorporation, which requires translocation of the product heteroduplex and separation of the downstream DNA duplex. Each single-nucleotide addition cycle can be divided into at least four steps. First, the substrate NTP binds to the polymerase in a pre-insertion mode, followed by a second step in which the NTP occupies the NTP-binding site (or N-site) and is base paired to the template nucleotide. The 3′ end of the RNA is in the priming site (or P-site) [8]. In step three, the phosphoryl transfer reaction produces pyrophosphate (PP_i) and the RNA transcript, which has been extended into the N-site by one nucleotide. In the final step, the elongated primer–template translocates relative to the active site metal ions; in this process, the 3′ end of the RNA moves from the N-site to the P-site, vacating the N-site for the next cycle of nucleotide addition.

Insights into NTP binding and the translocation process have been obtained from two additional, recently determined crystal structures of T7 RNAP elongation complexes [9^••]. One is the structure of T7 RNAP complexed with DNA containing a transcription bubble, an RNA transcript and a nonhydrolyzable ATP analog, αβ-methylene ATP, bound at the insertion site. The other structure is T7 RNAP complexed with the product of nucleotide insertion and PP_i that is in a pre-translocation state. These structures are in what is termed the ‘closed’ conformation, which differs from the ‘semi-open’ conformation of the post-translocation product state by a 22° rotation of a five-helix subdomain in the fingers domain. It is concluded that the release of PP_i results in both translocation and downstream strand separation [9^••].

Section snippets

Structural transition of T7 RNAP from initiation to the elongation phase

A comparison of the T7 RNAP structure in its initiation phase complex with its structure in the elongation phase complex (Figure 1) shows that portions of the enzyme, primarily the N-terminal domain, have undergone major conformational changes that alter its shape and tertiary structure 6.••, 7.••. These structural changes involve three different regions of the N-terminal domain, each undergoing a different kind of conformational alteration [6^••]. A six-helix domain rotates by 140° and

Comparison of T7 RNAP with multisubunit RNAP

Several similarities, as well as a few differences, are observed when the structures of T7 RNAP and its various substrate complexes are compared with structures recently determined for the multisubunit DNA-dependent RNAPs 12.••, 13.••, 14., 15.. The T7 RNAP elongation complex and the yeast pol II elongation complex have a similar angle (about 90°) between the axes of the downstream DNA and the heteroduplex, although the dihedral angles differ. Furthermore, the lengths of the heteroduplexes are

Structural basis of translocation by T7 RNAP

The elongation of RNA transcripts by T7 RNAP occurs through a four-step cycle of nucleotide addition, phosphoryl transfer and DNA translocation. A complete set of structures for all four steps of single-nucleotide addition is now available 5., 6.••, 7.••, 9.••. Comparison of the structures of T7 RNAP in the pre-translocation and post-translocation states shows a significant pivoting rotation of a helical subdomain in the fingers domain [9^••]. This large portion of the fingers domain (123 amino

Strand displacement

Comparison of the pre-translocation and post-translocation complex structures shows that fingers domain rotation (resulting from PP_i dissociation) is associated with the unwinding of the downstream duplex DNA by one base pair concomitant with translocation [9^••]. As domain rotation results in the primer terminus of the heteroduplex product moving from the N-site to the P-site, the downstream template strand is necessarily also translocated by 3.4 Å (Figure 4). Yin and Steitz [6^••] noted that the

Conclusions

Crystal structures of single-subunit T7 RNAP captured at various stages of transcription and the nucleotide incorporation cycle have provided insights into the functional properties of this RNA polymerase. The specific initiation of transcription is provided by a promoter-binding site that recognizes the promoter sequence. Promoter clearance is produced by a major conformational change in the N-terminal third of the protein. This change destroys the promoter-binding site and creates a tunnel

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

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of special interest
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of outstanding interest

References (18)

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A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription
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P. Cramer et al.
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Science
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There are more references available in the full text version of this article.

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Current Opinion in Structural Biology

Abstract

Introduction

Section snippets

Structural transition of T7 RNAP from initiation to the elongation phase

Comparison of T7 RNAP with multisubunit RNAP

Structural basis of translocation by T7 RNAP

Strand displacement

Conclusions

References and recommended reading

References (18)

A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription

J. Mol. Biol.

Structural basis of transcription: RNA polymerase II at 2.8 Angstrom resolution

Science

Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution

Cell

Protein-nucleic acid interactions in transcription: a molecular analysis

Annu. Rev. Biochem.

An integrated model of the transcription complex in elongation, termination, and editing

Science

Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter

Biochemistry

Processivity in early stages of transcription by T7 RNA polymerase

Biochemistry

Structure of a transcribing T7 RNA polymerase initiation complex

Science

Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase

Science

Cited by (51)

DNA–protein cross-links: Formidable challenges to maintaining genome integrity

Modeling macromolecular motions by x-ray-scattering-constrained molecular dynamics

T7 RNA polymerases backed up by covalently trapped proteins catalyze highly error prone transcription

Single-molecule studies of transcription: From one RNA polymerase at a time to the gene expression profile of a cell

Effects of viscogens on RNA transcription inside reovirus particles

Orthogonal gene expression in Escherichia coli