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.     

Physico-chemical study of complex formation of DNA with wild-type and mutant E. coli RNA polymerases. Recognition properties of beta-subunit

Complex formation of T₇ DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W 12) and its rifampicin-resistant mutant rpoB409 was studied. The rpoB409 mutant possesses a highly pleiotropic effect due to alteration in the RNA polymerase β-subunit structure. The two RNA polymerases have been previously shown to differ in gene selection during RNA synthesis on T₇ DNA. In this study it was found that the change in selective properties of the mutant RNA polymerase occurs during its interaction with DNA, the general ability of the enzyme to melt DNA being unaffected.     

INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase

RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase.     

A novel priming system for conjugal synthesis of an IncI alpha plasmid in recipients.

Summary Synthesis of DNA complementary to the transferred strand of an IncI plasmid has been shown previously to require DNA polymerase III. The possible involvement of the two defined priming proteins of Escherichia coli K12, RNA polymerase and primase, in initiating this conjugal DNA synthesis has been examined. Primase was inactivated using temperature-sensitive dnaG3 mutants and RNA polymerase was inhibited using rifampicin. When these two proteins were simultaneously inactivated in both parental strains, the average recipient synthesised at least one single-stranded equivalent of R144drd-3 before the rifampicin-treated donors lost the ability to transmit DNA. It is proposed that the product of a plasmid transfer gene is responsible for initiating this DNA synthesis in recipients. The results imply that this protein is supplied by the donors.     

真核基因启动子非依赖的功能转录延伸复合物的体外组装

真核生物RNA聚合酶Ⅱ的持续合成能力对基因转录过程中每一个阶段,包括启动子脱离、转录暂停、转录终止以及转录偶联DNA损伤修复过程的调节至关重要.在RNA聚合酶Ⅱ介导的转录延伸过程中,其和模板DNA及转录产物RNA紧密结合,形成一个非常稳定的延伸三维复合物(elongationcomplex,EC).此特征性“泡”状结构的形成是RNA聚合酶Ⅱ持续合成能力所必需的.在不依赖启动子及众多转录起始因子的条件下,利用人工合成的RNA与DNA寡核苷酸,在体外组装形成具有功能转录活性的延伸复合物.结果表明,长度为9个核苷酸的RNA与模板DNA形成的杂合分子对转录延伸复合物的形成是必需的,而非转录模板DNA链的加入导致最终活性转录“泡”状复合物的形成,并可转录形成与模板相关的转录产物,进一步通过在模板DNA的特定位置引入一个乙酰氧乙酰氨基芴修饰基团,可特异性地阻断转录延伸过程,从而显示该系统在研究真核基因转录及转录偶联DNA损伤修复机制中的潜在应用价值.     

H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter −10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter −10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.     

Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex. A neutron small angle scattering study

In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes. We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7. Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ. From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change. (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm. On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed. The main feature of this model is the positioning of RNA polymerase to only one side of the DNA.     

Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29

The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.     

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.