Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

1. Download : Download high-res image (168KB)
2. Download : Download full-size image

     

RNA Polymerase As a Molecular Machine

Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. It is unlikely that mechanical energy and mechanical devices such as power-stroke motor are used in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex, thus providing the directionality of the conveyance (Brownian ratchet mechanism). The following sequence of events: substrate binding fixation of a certain conformational state chemical reaction fixation of an alternative conformational state translocation (dissociation and downstream reassociation) of the product–template duplex is proposed as the principal scheme of the forward movement of RNA polymerase along the DNA template.     

Structure of the RNA‐directed RNA Polymerase from the cystovirus ϕ12

We have determined the structure of P2, the self‐priming RdRp from cystovirus ?12 in two crystal forms (A, B) at resolutions of 1.7 Å and 2.1 Å. Form A contains Mg²⁺ bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts‐mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo. Proteins 2013; 81:1479–1484. © 2013 Wiley Periodicals, Inc.     

Replication of double-stranded RNA in mycovirus from the plant pathogenic fungus, Fusarium solani

Abstract A mycovirus (named FusoV) from the plant pathogenic fungus Fusarium solani possessed two types of double-stranded (ds) RNA genome, designated Ml and M2. RNA-dependent RNA polymerase activity was detected in FusoV particle fractions. An in vitro RNA polymerase reaction using purified FusoV particles that was supplemented with NTPs revealed the synthesis of single-stranded (ss) RNA species and a subsequent formation of dsRNAs having the same size as Ml and M2. The ssRNA species synthesized in the first stage were proved to be of positive polarity (coding strand) for both M1 and M2 by dot blot hybridization analysis. These results suggest that FusoV genomic dsRNA replicates in a conservative manner.     

Revealing the DNA Unwinding Activity and Mechanism of Fork Reversal by RecG While Exposed to Variants of Stalled Replication-fork at Single-Molecular Resolution

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its ‘sliding back’ mode along the lagging daughter strand to be ‘active translocation and passive unwinding’. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.     

Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.     

用T7RNA聚合酶体外转录合成大鼠肝tRNA~(Ile)

采用PCR技术从rec M1 3mp1 8中扩增出 1 2 0bp的大鼠肝tRNAIle合成基因片段 ,经限制性内切酶BstNⅠ酶切后作为模板 ,利用T7RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNAIle基因 ,生成不含修饰碱基的tRNAIle,并对体外转录反应条件进行了优化 ,回收的tRNA产量可达DNA模板量的 4 0倍     

BRCA2 Regulates Transcription Elongation by RNA Polymerase II to Prevent R-Loop Accumulation

1. Download high-res image (192KB)
2. Download full-size image