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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription termination by RNA polymerase I (Pol I) is a stepwise process. First the elongating RNA polymerase is forced to pause by DNA-bound transcription termination factor (TTF-I). Then the ternary transcription complex is dissociated by PTRF, a novel factor that promotes release of both nascent transcripts and Pol I from the template. In this study we have investigated the ability of PTRF to liberate transcripts from ternary transcription complexes isolated from yeast and mouse. Using immobilized, tailed templates that contain terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we demonstrate that PTRF promotes release of terminated transcripts, irrespective of whether mouse Pol I has interacted with the murine termination factor TTF-I or its yeast homolog Reb1p. In contrast, mouse Pol I paused by the lac repressor remains bound to the template both in the presence and absence of PTRF. We demonstrate that PTRF interacts with the largest subunit of murine Pol I, with TTF-I and Reb1p, but not the lac repressor. The results imply that Pol I transcription termination in yeast and mouse is mediated by conserved interactions between Pol I, Reb1p/TTF-I and PTRF.
Mol Gen Genet 1999 Oct
PMID:Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse. 1058 39

Mammalian DNA polymerase beta functions in the base excision DNA repair pathway filling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of beta-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5'-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RMSD of the NMR conformers (residues 13-80) is 0.37 A for the backbone heavy atoms and 0.78 A for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5'-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Omega-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (R(ex)) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Omega-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.
J Mol Biol 2000 Feb 11
PMID:Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity. 1065 29

The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years. The base-selection (polymerase, or Pol) and editing (3'-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicative polymerases of other organisms, are primary determinants of the high fidelity of DNA synthesis in phage DNA replication. T4 gp43 is considered to be a member of the "B family" of DNA-dependent DNA polymerases (those resembling eukaryotic Pol alpha) because it exhibits striking similarities in primary structure to these enzymes. It has been extensively analyzed at the genetic, physiological, and biochemical levels; however, relationships between the in vivo properties of this enzyme and its physical structure have not always been easy to explain due to a paucity of structural data on the intact molecule. However, gp43 from phage RB69, a phylogenetic relative of T4, was crystallized and its structure solved in a complex with single-stranded DNA occupying the Exo site, as well as in the unliganded form. Analyses with these crystals, and crystals of a T4 gp43 proteolytic fragment harboring the Exo function, are opening new avenues to interpret existing biological and biochemical data on the intact T4 enzyme and are revealing new aspects of the microanatomy of gp43 that can now be explored further for functional significance. We summarize our current understanding of gp43 structure and review the physiological roles of this protein as an essential DNA-binding component of the multiprotein T4 DNA replication complex and as a nucleotide-sequence-specific RNA-binding translational repressor that controls its own biosynthesis and activity in vivo. We also contrast the properties of the T4 DNA replication complex to the functionally analogous complexes of other organisms, particularly Escherichia coli, and point out some of the unanswered questions about gp43 and T4 DNA replication.
Prog Nucleic Acid Res Mol Biol 2000
PMID:DNA polymerase of the T4-related bacteriophages. 1069 7

The molecular basis of human characteristics is an intriguing but an unresolved problem. Human characteristics cover a broad spectrum, from the obvious to the abstract. Obvious characteristics may include morphological features such as height, shape, and facial form. Abstract characteristics may be hidden in processes that are controlled by hormones and the human brain. In this review we examine exaggerated characteristics presented as syndromes. Specifically, we focus on human genes that encode transcription factors to examine morphological, immunological, and hormonal anomalies that result from deletion, insertion, or mutation of genes that regulate transcription by RNA polymerase II (the Pol II genes). A close analysis of abnormal phenotypes can give clues into how sequence variations in regulatory genes and changes in transcriptional control may give rise to characteristics defined as complex traits.
Prog Nucleic Acid Res Mol Biol 2000
PMID:Syndromes associated with Homo sapiens pol II regulatory genes. 1069 10

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.
Mol Cell Biol 2000 Jun
PMID:Multiple signals regulate GAL transcription in yeast. 1080 31

In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.
Mol Cell Biol 2000 Jun
PMID:Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells. 1082 98

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.
J Mol Biol 2000 May 26
PMID:Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1. 1086 Jul 20

Herpes simplex virus DNA polymerase is a heterodimer composed of a catalytic subunit, Pol, and an unusual processivity subunit, UL42, which, unlike processivity factors such as PCNA, directly binds DNA. The crystal structure of a complex of the C-terminal 36 residues of Pol bound to residues 1-319 of UL42 reveals remarkable similarities between UL42 and PCNA despite contrasting biochemical properties and lack of sequence homology. Moreover, the Pol-UL42 interaction resembles the interaction between the cell cycle regulator p21 and PCNA. The structure and previous data suggest that the UL42 monomer interacts with DNA quite differently than does multimeric toroidal PCNA. The details of the structure lead to a model for the mechanism of UL42, provide the basis for drug design, and allow modeling of other proteins that lack sequence homology with UL42 or PCNA.
Mol Cell 2000 Feb
PMID:The crystal structure of an unusual processivity factor, herpes simplex virus UL42, bound to the C terminus of its cognate polymerase. 1088 68

Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.
Mol Cell 2000 Feb
PMID:The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. 1088 76

Modulation of gene expression by catalytic RNA requires accessible ribozyme cleavage sites in the target mRNA, and accessibility is determined by the secondary and tertiary structure of the target RNA, as affected by its interactions with cellular proteins. As we previously reported, an oligonucleotide-scanning approach using antisense oligonucleotides can be used to determine RNA accessibility in cell extracts. To test whether this method can be used to improve selection of ribozyme target sites, we designed ribozymes corresponding to the sites identified by oligonucleotide scanning and have evaluated their catalytic activities, first in cell extracts and then in transduced cell lines. As a target we used the mRNA of murine DNA (cytosine-5)-methyltransferase 1 (MTase). For intracellular studies, the ribozyme genes were inserted downstream of a Pol III tRNAVAL promoter, which in turn was cloned in the U3 region of a retroviral vector. We find that the efficiency of the ribozymes both in cell extracts and in vivo corresponds with the relative effectiveness predicted by the oligonucleotide-scanning assay. The best ribozyme causes a 70-80% reduction in the MTase mRNA levels in NIH 3T3 cells that are stably transduced with the retroviral constructs. This reduction in mRNA levels is accompanied by a small decrease in the methylation of repetitive intercisternal A particle DNA elements. Ribozyme expression also increased several-fold the reactivation frequency of a methylation-silenced green fluorescent protein (GFP) transgene. Both the reduction in methylation and reactivation of GFP were roughly equivalent to the effects obtained by treating NIH 3T3 cells with 2.5 microM 5-azacytidine, which gives an effect of about 10% of maximum. These results confirm the validity of the cell extract approach for ribozyme site selection and provide a potentially useful ribozyme for future study of DNA methyltransferase function.
Mol Ther 2000 Jul
PMID:Oligonucleotide scanning of native mRNAs in extracts predicts intracellular ribozyme efficiency: ribozyme-mediated reduction of the murine DNA methyltransferase. 1089 25


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