Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phages lambda and HK022 express proteins N and Nun, respectively, each of which acts with a number of Escherichia coli host Nus factors at lambda NUT RNA sites, to influence transcription elongation. The lambda nut sites, nearly identical sequences located downstream of the early promoters, pL and pR, were first identified as cis-acting signals required for the action of N in forming termination-resistant transcription complexes. Surprisingly, the Nun protein, resembling N and expressed by another lambdoid phage, HK022, also acts with Nus proteins to terminate specifically transcription initiating at pL and pR near the lambda nut sites. Based on structural considerations of the amino acid sequences, we have constructed nine hybrid N-nun genes and used these hybrids to identify functional regions of the N and Nun proteins. Three classes of hybrid gene products were identified: (1) those that, like N, support antitermination, (2) those that, like Nun, terminate transcription, and (3) those that block N action but do not terminate transcription. We find that, similar to N, the amino-terminal portion of Nun is involved in RNA recognition. The more carboxy portions influence transcription elongation, antitermination (N) and termination (Nun). Depending on the derivations of the more carboxy regions, hybrids with either the N or Nun amino portions support either termination or antitermination. The activity of a hybrid protein may be influenced by the host strain depending on the nature of the rpoC locus, a locus encoding the beta' subunit of RNA polymerase. One of the hybrid proteins blocks antitermination when the rpoC locus is wild-type. The same hybrid in the presence of the rpoC100 mutation, which alters the beta' subunit, has antitermination activity. This result supports the argument that the beta' subunit plays an essential role in determining the progress of transcription elongation.
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PMID:Identification of functional regions of the Nun transcription termination protein of phage HK022 and the N antitermination protein of phage lambda using hybrid nun-N genes. 863 63

Coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage-encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination-resistant form. Expression of lambda late genes is regulated by the phage-encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination-resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins. (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination-resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non-template DNA strand. (iv) A host-encoded inhibitor of N antitermination appears to act through the NUT site as well as with the alpha subunit of RNA polymerase, and is antagonized by NusB protein.
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PMID:Transcription antitermination: the lambda paradigm updated. 870 39

Gene expression in lambdoid phages in part is controlled by transcription antitermination. For most lambdoid phages, maximal expression of delayed early genes requires an RNA polymerase modified by the phage N and host Nus proteins at RNA NUT sites. The NUT sites (NUTL and NUTR) are made up of three elements: BOXA, BOXB and an intervening spacer sequence. We report on N antitermination in H-19B, a lambdoid phage carrying shiga toxin 1 genes. H-19B N requires NusA, but not two other host factors required by lambda N, NusB and ribosomal protein S10. The H-19B NUT site BOXA is not required, whereas the BOXB is required for N action. H-19B nut sites have dyad symmetries in the spacer regions that are not in other nut sites. Changes in one arm of the dyad symmetry inactivate the NUT RNA. Compensating changes increasing the number of mutant nucleotides but restoring dyad symmetry restore activity. Deletion of the sequences encoding the dyad symmetry has little effect. Thus, the specific nucleotides composing the dyad symmetry seem relatively unimportant. We propose that the RNA stem-loop structure, called the 'reducer', by sequestering nucleotides from the linear RNA brings into proximity sites on either side of the dyad symmetry that contribute to forming an active NUT site.
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PMID:N-mediated transcription antitermination in lambdoid phage H-19B is characterized by alternative NUT RNA structures and a reduced requirement for host factors. 1112 80

The E. coli NusA transcription elongation protein (NusA(Ec)), identified because of its requirement for transcription antitermination by the N protein, has an Arg-rich S1 RNA-binding domain. A complex of N and NusA with other host factors binding at NUT sites in the RNA renders RNA polymerase termination-resistant. An E. coli haploid for nusA944, having nine different codons replacing four normally found in the Arg-rich region, is defective in support of N action. Another variant, haploid for the nusAR199A allele, with a change in a highly conserved Arg codon in the S1 domain, effectively supports N-mediated antitermination. However, nusAR199A is recessive to nusA944, while nusA(Ec) is dominant to nusA944 for support of N-mediated antitermination, suggesting a competition between NusA944 and NusAR199A during complex formation. Complex formation with the variant NusA proteins was assessed by mobility gel shifts. NusAR199A, unlike NusA(Ec) and NusA944, fails to form a complex with N and NUT RNA. However, while NusAR199A, like wild-type NusA, forms an enlarged complex with NUT RNA, N, RNA polymerase, and other host proteins required for efficient N-mediated antitermination, NusA944 does not form this enlarged complex. Consistent with the in vivo results, NusA944 prevents NusAR199A but not NusA(Ec) from forming the enlarged complex. The simplest conclusion from these dominance studies is that in the formation of the complete active antitermination complex in vivo, NusA and N binding to the newly synthesized NUT RNA precedes addition of the other factors. Alternative less effective routes to the active complex that allows bypass of this preferred pathway may also exist.
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PMID:Interactions of an Arg-rich region of transcription elongation protein NusA with NUT RNA: implications for the order of assembly of the lambda N antitermination complex in vivo. 1141 35

The N-terminal arginine-rich motif of phage HK022 Nun protein binds to NUT sequences in phage lambda nascent transcripts and induces transcription termination. Interactions between the Nun C terminus and RNA polymerase as well as the DNA template are required for termination. We have isolated Nun C-terminal point and deletion mutants that are unable to block transcription. The mutants bind NUT RNA and inhibit translation of the lambda N gene. Thus HK022 excludes lambda both by terminating transcription on the phage chromosome and by preventing translation of the essential lambda N gene. Like N autoregulation, translation repression by Nun requires host RNaseIII deficiency (rnc) or a mutation in the RNaseIII processing site (rIII) located between NUTL and the beginning of the N coding sequence. Our data support the idea that Nun bound at NUTL causes steric interference with ribosome attachment to the nearby N coding sequence. Two models, Nun acting alone or in complex with host proteins, are discussed.
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PMID:Phage HK022 Nun protein represses translation of phage lambda N (transcription termination/translation repression). 1268 30

Bacteriophage lambda N and bacterial Nus proteins together with a unique site NUT in the leader of the early viral N gene transcript bind RNA polymerase (RNAP) and form a highly processive antitermination complex; N bound at NUT also represses N translation. In this study, we investigate whether N and NUT cause N translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the N-modified transcription complex for their effect on N-mediated translation repression. We show that nus and nut mutations that in combination destabilize multiple interactions in the antitermination complex prevent N-mediated translation repression. Likewise, transcription of the nut-N region by T7 RNAP, which does not lead to the assembly of an effective antitermination complex when N is supplied, eliminates translation repression. We also demonstrate that a unique mutant beta subunit of RNAP reduces N-mediated translation repression, and that overexpression of transcription factor NusA suppresses this defect. We conclude that the N-modified RNAP transcription complex is necessary to repress N translation.
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PMID:Translation repression by an RNA polymerase elongation complex. 1525 95

The N protein of phage lambda acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the lambda early P(R) promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P(R), at positions -14 and -45 (yielding P(R-GA)), reduces effectiveness of N modification, while an additional mutation at position -30 (yielding P(R-GCA)) suppresses this effect. In vivo, the level of P(R-GA)-directed transcription was twice as great as the wild-type level, while transcription directed by P(R-GCA) was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P(R-GA) in vitro was roughly one-third the rate for wild-type P(R). We ascribe this apparent discrepancy to an effect of the mutations in P(R-GCA) on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.
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PMID:Evidence that the promoter can influence assembly of antitermination complexes at downstream RNA sites. 1651 52

The bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (CTD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other CTD kinases are inactive. Phosphorylation of the CTD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II CTD Ser2 kinase implicates it as a regulator of eukaryotic transcription.
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PMID:BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. 2250 28

BET proteins have recently become recognized for their role in a broad range of cancers and are defined by the presence of two acetyl-histone reading bromodomains and an ET domain. This family of proteins includes BRD2, BRD3, BRD4, and BRDT. BRD4 is the most-studied BET protein in cancer, and normally serves as an epigenetic reader that links active chromatin marks to transcriptional elongation through activation of RNA polymerase II. The role of BRD3 and BRD4 first became known in cancer as mutant oncoproteins fused to the p300-recruiting NUT protein in a rare aggressive subtype of squamous cell cancer known as NUT midline carcinoma (NMC). BET inhibitors are acetyl-histone mimetics that specifically bind BET bromodomains, competitively inhibiting its engagement with chromatin. The antineoplastic effects of BET inhibitors were first demonstrated in NMC and have since been shown to be effective at inhibiting the growth of many different cancers, particularly acute leukemia. BET inhibitors have also been instrumental as tool compounds that have demonstrated the key role of BRD4 in driving NMC and non-NMC cancer growth. Many clinical trials enrolling patients with hematologic and solid tumors are ongoing, with encouraging preliminary findings. BET proteins BRD2, BRD3, and BRD4 are expressed in nearly all cells of the body, so there are concerns of toxicity with BET inhibitors, as well as the development of resistance. Toxicity and resistance may be overcome by combining BET inhibitors with other targeted inhibitors, or through the use of novel BET inhibitor derivatives.
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PMID:Small-Molecule Targeting of BET Proteins in Cancer. 2745 Nov 23

Delineating how chromosomes fold at length scales beyond one megabase remains obscure relative to smaller-scale folding into TADs, loops, and nucleosomes. We find that rather than simply unfolding chromatin, histone hyperacetylation results in interactions between distant genomic loci separated by tens to hundreds of megabases, even in the absence of transcription. These hyperacetylated "megadomains" are formed by the BRD4-NUT fusion oncoprotein, interact both within and between chromosomes, and form a specific nuclear subcompartment that has elevated gene activity with respect to other subcompartments. Pharmacological degradation of BRD4-NUT results in collapse of megadomains and attenuation of the interactions between them. In contrast, these interactions persist and contacts between newly acetylated regions are formed after inhibiting RNA polymerase II initiation. Our structure-function approach thus reveals that broad chromatin domains of identical biochemical composition, independent of transcription, form nuclear subcompartments, and also indicates the potential of altering chromosome structure for treating human disease.
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PMID:Chromatin Hyperacetylation Impacts Chromosome Folding by Forming a Nuclear Subcompartment. 3228 35


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