共查询到20条相似文献,搜索用时 31 毫秒
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V Gopal L G Brieba R Guajardo W T McAllister R Sousa 《Journal of molecular biology》1999,290(2):411-431
We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modifications on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid. 相似文献
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Ma K Temiakov D Jiang M Anikin M McAllister WT 《The Journal of biological chemistry》2002,277(45):43206-43215
To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid approximately 9 bp upstream from the active site (at -9) it interacts with a region within the specificity loop (residues 744-750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172-191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination. 相似文献
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Alfredo J. Hernandez Seung-Joo Lee Noah J. Thompson Jack D. Griffith Charles C. Richardson 《The Journal of biological chemistry》2022,298(6)
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer. 相似文献
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Conte MR Kelly G Babon J Sanfelice D Youell J Smerdon SJ Proud CG 《Biochemistry》2006,45(14):4550-4558
Eukaryotic initiation factor 5 (eIF5) plays multiple roles in translation initiation. Its N-terminal domain functions as a GTPase-activator protein (GAP) for GTP bound to eIF2, while its C-terminal region nucleates the interactions between multiple translation factors, including eIF1, which acts to inhibit GTP hydrolysis or P(i) release, and the beta subunit of eIF2. These proteins and the events in which they participate are critical for the accurate recognition of the correct start codon during translation initiation. Here, we report the three-dimensional solution structure of the N-terminal domain of human eIF5, comprising two subdomains, both reminiscent of nucleic-acid-binding modules. The N-terminal subdomain contains the "arginine finger" motif that is essential for GAP function but which, unusually, resides in a partially disordered region of the molecule. This implies that a conformational reordering of this portion of eIF5 is likely to occur upon formation of a competent complex for GTP hydrolysis, following the appropriate activation signal. Interestingly, the N-terminal subdomain of eIF5 reveals an alpha/beta fold structurally similar to both the archaeal orthologue of the beta subunit of eIF2 and, unexpectedly, to eIF1. These results reveal a novel protein fold common to several factors involved in related steps of translation initiation. The implications of these observations are discussed in terms of the mechanism of translation initiation. 相似文献
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Kim YC Russell WK Ranjith-Kumar CT Thomson M Russell DH Kao CC 《The Journal of biological chemistry》2005,280(45):38011-38019
Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis. 相似文献
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Dematin is an actin binding protein from the junctional complex of the erythrocyte cytoskeleton. The protein has two actin binding sites and bundles actin filaments in vitro. This actin bundling activity is reversibly regulated by phosphorylation in the carboxyl terminal "headpiece" domain (DHP). DHP is a typical villin-type headpiece actin binding motif and contains a flexible N-terminal loop and an alpha-helical C-terminal subdomain that is phosphorylated at Ser74. The NMR structure of a Ser74-to-Glu mutant (DHPs74e) closely mimics the conformation of phosphorylated DHP. The negative charge at Ser74 does not alter the conformation of the C-terminal subdomain, but attracts the N-terminal loop toward the C terminus, changing the orientation of the N-terminal subdomain. NMR relaxation studies also indicate reduced mobility in the N-terminal loop in DHPs74e. Thus, phosphorylation in DHP serves as a switch controlling the conformational state of DHP and the actin bundling activity of dematin. 相似文献
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Dutartre H Boretto J Guillemot JC Canard B 《The Journal of biological chemistry》2005,280(8):6359-6368
Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation. 相似文献
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Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure
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UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site. 相似文献
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The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis. 相似文献
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