Abstract:
:Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
San Filippo J,Lambowitz AMdoi
10.1016/s0022-2836(02)01147-6keywords:
subject
Has Abstractpub_date
2002-12-13 00:00:00pages
933-51issue
5eissn
0022-2836issn
1089-8638pii
S0022283602011476journal_volume
324pub_type
杂志文章abstract::The repertoire of naturally occurring protein structures is usually characterised in structural terms at the domain level by their constituent folds. As structure is acknowledged to be an important stepping stone to the understanding of protein function, an appreciation of how individual domain interactions are built ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2004.11.011
更新日期:2005-02-04 00:00:00
abstract::The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2005.02.034
更新日期:2005-05-06 00:00:00
abstract::RNA localization is an important means of post-transcriptional regulation of gene expression in many eukaryotic cell types. In neurons, select RNAs are delivered to postsynaptic dendritic microdomains, a mechanism that is considered a key underpinning in the administration of long-term synaptic plasticity. BC1 RNA is ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2005.11.090
更新日期:2006-03-10 00:00:00
abstract::Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After hea...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(84)90461-3
更新日期:1984-08-05 00:00:00
abstract::Opsins are widespread photoreceptor proteins involved in a diversity of light-driven processes in all major taxa that use the apocarotenoid retinal as a light-absorbing prosthetic group. Proteins from the opsin family are also found in filamentous fungi, but no function has been attributed to them. The fungus Fusarium...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2009.01.057
更新日期:2009-03-20 00:00:00
abstract::Residue-specific potentials between pairs of side-chains and pairs of side-chain-backbone interaction sites have been generated by collecting radial distribution data for 302 protein structures. Multiple atomic interactions have been utilized to enhance the specificity and smooth the distance-dependence of the potenti...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0758
更新日期:1997-02-14 00:00:00
abstract::Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of AT...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.3467
更新日期:2000-02-18 00:00:00
abstract::The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed indepen...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2001.5123
更新日期:2001-11-16 00:00:00
abstract::Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabol...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(02)01235-4
更新日期:2003-01-17 00:00:00
abstract::Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1998.1672
更新日期:1998-04-24 00:00:00
abstract::The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 A resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2010.05.043
更新日期:2010-08-06 00:00:00
abstract::We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and induci...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2009.10.030
更新日期:2010-01-15 00:00:00
abstract::Ferredoxin-NAD(P)(+) oxidoreductase (FNR) catalyzes the reduction of NAD(P)(+) to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2010.06.024
更新日期:2010-08-20 00:00:00
abstract::Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibr...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2018.03.022
更新日期:2018-05-11 00:00:00
abstract::A detailed phylogenetic analysis for mammalian members of the family Herpesviridae, based on molecular sequences is reported. Sets of encoded amino acid sequences were collected for eight well conserved genes that are common to mammalian herpesviruses. Phylogenetic trees were inferred from alignments of these sequence...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1995.0152
更新日期:1995-03-31 00:00:00
abstract::To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cp...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2020.03.033
更新日期:2020-05-01 00:00:00
abstract::The secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% alpha-helix and 32 to 33% beta-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1993.1442
更新日期:1993-08-05 00:00:00
abstract::Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2014.09.024
更新日期:2015-05-08 00:00:00
abstract::Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of ...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2014.09.023
更新日期:2014-12-12 00:00:00
abstract::As part of an overall effort to map the energetic landscape of the base excision repair pathway, we report the first thermodynamic characterization of repair enzyme binding to lesion-containing duplexes. Isothermal titration calorimetry (ITC) in conjunction with spectroscopic measurements and protease protection assay...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(03)00365-6
更新日期:2003-05-16 00:00:00
abstract::The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteas...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2008.12.081
更新日期:2009-02-27 00:00:00
abstract::UDP -N- acetylmuramoyl- L -alanine: D -glutamate (MurD) ligase catalyses the addition of d -glutamate to the nucleotide precursor UDP -N- acetylmuramoyl- L -alanine (UMA). The crystal structures of three complexes of Escherichia coli MurD with a variety of substrates and products have been determined to high resolutio...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.2800
更新日期:1999-06-11 00:00:00
abstract::We have studied the reversible dissociation of core size DNA from chicken erythrocyte nucleosome core particles in solutions containing 0 X 1 M to 0 X 6 M-NaCl. Dissociation increases with increasing NaCl concentration, increasing temperature and decreasing particle concentration. At high particle concentrations, no f...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(84)90383-8
更新日期:1984-06-15 00:00:00
abstract::Solid-state 2H nuclear magnetic resonance line shapes have been obtained from folded films of oriented Li-DNA molecules with the purine bases selectively labeled with deuterium at the 8-position. From line shape simulations, the static base tilts as well as the anisotropic motional amplitudes were determined as a func...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(88)90461-5
更新日期:1988-07-20 00:00:00
abstract::Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie crucial developmental processes such as the fo...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2011.03.013
更新日期:2011-05-13 00:00:00
abstract::Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Ta...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2010.10.052
更新日期:2011-01-07 00:00:00
abstract::The enzyme encoded by the bgIA gene of Bacillus polymyxa, a type I beta-glucosidase belonging to family I of glycosyl hydrolases, has been purified to homogeneity from an Escherichia coli culture which overexpressed the gene, and crystallized. The crystals, which diffract to 3.0 A resolution, belong to the orthorhombi...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1994.1441
更新日期:1994-07-15 00:00:00
abstract::Botulinum neurotoxin (BoNT) is a category A toxin that has been classified within seven serotypes, designated A-G. Recently, it has been discovered that sequence variability occurs in BoNTs produced by serotype A (BoNT/A) variant strains, designated as subtypes A1 and A2, which have significantly different antibody-bi...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2006.07.040
更新日期:2006-09-29 00:00:00
abstract::The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2009.11.009
更新日期:2010-01-22 00:00:00
abstract::DNA structures bound to transcription factors are studied by using the crystal coordinates of complexes. Structural characteristics which are found at the sites where a protein secondary structure, an alpha-helix or a beta-sheet binds, can be understood in terms of fitting of the concave surface of the DNA major groov...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1996.0055
更新日期:1996-02-09 00:00:00