Structure Of T7 RNA Polymerase Research Articles

Visualizing polynucleotide polymerase machines at work.

The structures of T7 RNA polymerase (T7 RNAP) captured in the initiation and elongation phases of transcription, that of phi29 DNA polymerase bound to a primer protein and those of the multisubunit RNAPs bound to initiating factors provide insights into how these proteins can initiate RNA synthesis and synthesize 6-10 nucleotides while remaining bound to the site of initiation. Structural insight into the translocation of the product transcript and the separation of the downstream duplex DNA is provided by the structures of the four states of nucleotide incorporation. Single molecule and biochemical studies show a distribution of primer terminus positions that is altered by the binding of NTP and PP(i) ligands. This article reviews the insights that imaging the structure of polynucleotide polymerases at different steps of the polymerization reaction has provided on the mechanisms of the polymerization reaction. Movies are shown that allow the direct visualization of the conformational changes that the polymerases undergo during the different steps of polymerization.

Unravelling the Mechanism of RNA-Polymerase Forward Motion by Using Mechanical Force

Polymerases form a class of enzymes that act as molecular motors as they move along their nucleic acid substrate during catalysis, incorporating nucleotide triphosphates at the end of the growing chain and consuming chemical energy. A debated issue is how the enzyme converts chemical energy into motion [J. Gelles and R. Landick, Cell 93, 13 (1998)]. In a single molecule assay, we studied how an opposing mechanical force affects the translocation rate of T7 RNA polymerase. Our measurements show that force acts as a competitive inhibitor of nucleotide binding. This result is interpreted in the context of possible models, and with respect to published crystal structures of T7 RNA polymerase. The transcribing complex appears to utilize only a small fraction of the energy of hydrolysis to perform mechanical work, with the remainder being converted to heat.

Probing the interaction of T7 RNA polymerase with promoter.

Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellent candidate for studying structural aspects of transcription, because unlike the eucaryotic and bacterial RNA polymerases, it is a single subunit enzyme and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying transcription using this enzyme is that the high-resolution crystal structure of T7 RNA polymerase has been solved. However, a cocrystal structure of the polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promoter with T7 RNA polymerase. We constructed promoters that were substituted with the photo-cross-linkable nucleotide 5-iodo uracil at every dT in the promoter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In the melting domain, the strongest contacts occurred at -3 and at -1 on the template strand while very weak cross-linking was seen at -2 and at -4 on the nontemplate strand. In the binding domain, the strongest contacts were seen at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-linking was poor in the intervening region between the binding and the melting domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding. For a systematic study designed to probe the nature of base-specific interactions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate > citrate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contacts, we observed that at -2, -4, and -17 the contacts had a hydrophobic component, while at many other positions there was no significant effect, suggesting that the contacts in the promoter-polymerase complexes were predominantly ionic but at certain positions nonionic interactions also existed. To localize a specific interaction in the melting domain, we proteolyzed the cross-linked T7 RNAP and analyzed the fragments using gel electrophoresis, mass spectrometry, and amino acid composition. High-resolution mapping indicated that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.

Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.

The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold.

Identification of the template-binding cleft of T7 RNA polymerase as the site for promoter binding by photochemical cross-linking with psoralen.

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether. We apply this method for the interaction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, and fingers domains. It was proposed that template DNA binds in the cleft. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linked tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal structure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526-5538] we used site-specific psoralen furan-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polymerase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to identify the location of this peptide on the T7 RNA polymerase primary sequence. In the present work we have mapped this peptide on the 3-D structure of T7 RNA polymerase. This peptide maps in the fingers domain of the polymerase. On the basis of a comparison of the map positions of peptides that cross-linked to either promoter DNA or single-stranded oligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-stranded DNA. Whereas the cleft of the polymerase is the seat of double-stranded promoter binding, the fingers domain may be used by the polymerase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alternatively, the single-stranded oligo binding site may be an RNA product-binding site during transcription. The photochemical techniques we have developed [Sastry et al. (1993) Biochemistry 32, 5526-5538; this work] can be applied to other DNA-protein complexes to map DNA-binding domains.

Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination

Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (~90%) of these initiation events lead to synthesis of dinucleotides only. The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other nonpromoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation. Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP. The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs). Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator. Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP. These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template. The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain. This interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex. Transcription termination is a reversal of the abortive to processive switch; hairpin formation disrupts the RNAP-RNA interaction and concomitantly destabilizes the processive conformation. In other words, the nascent RNA is proposed to act as the effector ligand of a non-formal allosteric transition in T7 RNAP that mediates both the switch from abortive to processive transcription and transcription termination.