Abstract:
:DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Bailey MF,van der Schans EJ,Millar DPdoi
10.1016/j.jmb.2003.11.023keywords:
subject
Has Abstractpub_date
2004-02-20 00:00:00pages
673-93issue
3eissn
0022-2836issn
1089-8638pii
S0022283603014190journal_volume
336pub_type
杂志文章abstract::The structures of transition states and intermediates in protein folding may be analysed by protein engineering methods that remove simple interactions that stabilize the folded state. We have now extended the range and reliability of the procedure by using the COSMIC (Combination of Sequential Mutant Interaction Cycl...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(91)90852-w
更新日期:1991-05-05 00:00:00
abstract::Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated. Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H. halobium single rRNA operon. Three types of mutations were found in pactamycin resistant cells, A694...
journal_title:Journal of molecular biology
pub_type: 杂志文章,评审
doi:10.1006/jmbi.1997.1387
更新日期:1997-11-21 00:00:00
abstract::Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E. coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes o...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(88)90573-6
更新日期:1988-11-20 00:00:00
abstract::Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(87)90255-5
更新日期:1987-04-20 00:00:00
abstract::The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that lead to cardiac muscle contraction. The cardiac N-terminal domain of TnC consists o...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2011.08.049
更新日期:2011-10-28 00:00:00
abstract::The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternat...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2012.06.032
更新日期:2012-10-12 00:00:00
abstract::Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(02)00732-5
更新日期:2002-08-30 00:00:00
abstract::l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD(+)-dependent enzyme in the uronate cycle but also as a taxon-specific lambda-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer cons...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2010.06.069
更新日期:2010-09-03 00:00:00
abstract::The great diversity of RNA biological functions has led to widespread interest in RNA structure. Advances in synthetic and spectroscopic techniques have recently allowed the extension of NMR methods of structure determination to RNA structures of significant size and increased biological significance. However, it has ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0855
更新日期:1997-03-28 00:00:00
abstract::Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connect...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2000.3712
更新日期:2000-05-19 00:00:00
abstract::The temperature dependence of processing of precursor tRNA(Gly) (ptRNA(Gly)) variants carrying a single 2'-OCH(3) or locked nucleic acid (LNA) modification at G+1 by Escherichia coli endoribonuclease P RNA was studied at rate-limiting chemistry. We show, for the first time, that these ribose modifications at nucleotid...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.03.042
更新日期:2008-05-23 00:00:00
abstract::Interleukin (IL)-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cy...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.07.051
更新日期:2008-10-17 00:00:00
abstract::We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been describ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2006.06.011
更新日期:2006-07-28 00:00:00
abstract::Plasmids were constructed in which a HindIII fragment of rDNA (6.4 x 10(3) base-pairs) was inserted into vectors pGEM-1 and 2 in both orientations. The DNA insert encoded the yeast 35 S precursor rRNA beginning 180 bases upstream from the 5' end of the mature 18 S rRNA and ending 289 bases beyond the 3' end of the mat...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(90)90353-N
更新日期:1990-01-20 00:00:00
abstract::The rate of synthesis and the concentrations of a variety of messenger RNA sequences have been compared between adult mouse liver cells and cells in other adult tissues, and between cells in fetal and neonatal liver. The sequences were distinguished as "liver-specific" or "common" in a previous report, where liver was...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(84)90304-8
更新日期:1984-10-15 00:00:00
abstract::Reverse transcriptases (RTs) encoded by a wide range of mobile retroelements have had a major impact on the structure and function of genomes. Among the most abundant elements in eukaryotes are the non long terminal repeat (LTR) retrotransposons. Here we compare the dNTP concentration requirements and error rates of t...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2011.02.015
更新日期:2011-04-15 00:00:00
abstract::Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K(d) values that vary between 10(-4) and 10(-14) M, characteristics that are explained by a 'dual-recognition' mechanism. In thi...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.03.055
更新日期:2008-06-13 00:00:00
abstract::We show that all minor coat proteins of phage f1 are integral inner membrane proteins prior to assembly. Hence all phage structural and morphogenetic proteins are localized in the membrane of the infected cell, consistent with models of phage assembly in which morphogenesis is coincident with phage extrusion. Our data...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1995.0393
更新日期:1995-07-21 00:00:00
abstract::Heme is an essential prosthetic group of proteins involved in oxygen transport, energy metabolism and nitric oxide production. ALAS1 (5-aminolevulinate synthase) is the rate-limiting enzyme in heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, ...
journal_title:Journal of molecular biology
pub_type: 杂志文章,收录出版
doi:10.1016/j.jmb.2009.03.024
更新日期:2009-05-01 00:00:00
abstract::The increased incidence of bacterial resistance to available antibiotics represents a major global health problem and highlights the need for novel anti-infective therapies. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. AMPs are versatile, have almost unlimited sequence sp...
journal_title:Journal of molecular biology
pub_type: 杂志文章,评审
doi:10.1016/j.jmb.2018.12.015
更新日期:2019-08-23 00:00:00
abstract::The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form orde...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(83)80015-1
更新日期:1983-08-05 00:00:00
abstract::Halobacterium salinarium is a chemo- and phototactic archaeon whose signal transduction pathway includes the classical two-component system made up of CheA and CheY. Deletion analysis of the che operon in H. salinarium has been undertaken. Following the removal of the entire operon, the importance of each of the four ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0267
更新日期:1996-05-17 00:00:00
abstract::The structure of active site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor was determined in the presence of coenzyme NAD+ at 1.88 A resolution with a final R-factor of 0.175. The structure refinement was carried out on the basis of the structure of holo-GAPDH at 2.0 A resoluti...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.2628
更新日期:1999-04-09 00:00:00
abstract::We identified the predominant 5' ends of an mRNA in Escherichia coli to the exact nucleotides. There are four such ends of lac mRNA in fully induced cells. About 70% of the molecules have the reported major in vitro end, A-A-U-U-G (at +1), which is located 38 nucleotides before the A-U-G translation start. Another 15%...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(85)90342-0
更新日期:1985-03-20 00:00:00
abstract::Among the unexpected chemistries that can be catalyzed by nucleic acid enzymes is photochemistry. We have reported the in vitro selection of a small, cofactor-independent deoxyribozyme, UV1C, capable of repairing thymine dimers in a DNA substrate, most optimally with light at a wavelength of >300 nm. We hypothesized t...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2006.10.062
更新日期:2007-02-02 00:00:00
abstract::The Escherichia coli RNA degradosome is a multienzyme assembly that functions in transcript turnover and maturation of structured RNA precursors. We have developed a procedure to reconstitute the RNA degradosome from recombinant components using modular coexpression vectors. The reconstituted assembly can be purified ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.07.059
更新日期:2008-10-17 00:00:00
abstract::A gene encoding a major entomocidal polypeptide from Bacillus thuringiensis subspecies kyushuensis delta-endotoxin crystals (CytB) was cloned into Escherichia coli and sequenced. The deduced amino acid sequence gave a predicted molecular mass of 29,236 Da and showed 39% identity and 70% similarity with the 27,371 Da C...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1993.1037
更新日期:1993-01-20 00:00:00
abstract::We describe new tests, of general application, for deciding whether two proteins or DNA sequences are significantly homologous, in cases where the relationship is neither evidently true nor evidently false. Ralston and Bishop's comparison of the c-myc oncogene with the adenovirus E1a protein is discussed as an example...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(85)90181-0
更新日期:1985-09-05 00:00:00
abstract::Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers we...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2010.11.014
更新日期:2011-01-21 00:00:00
abstract::K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10t...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2016.11.008
更新日期:2017-02-17 00:00:00