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)

The compound 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl)adenine 5'-monophosphate is an inhibitor (Ki = 330 microM) of the initiation binding site of the DNA-dependent RNA polymerase derived from Escherichia coli. The alpha-32P derivative of this photo-labile compound is used to derivatize a site on the sigma subunit of the holoenzyme (E sigma) using either T7 delta D111 or poly[d(A-T)] as a DNA template. The incorporation of the 32P label into the sigma subunit could be prevented by the addition of either 5'-AMP or 5'-ATP. The results are suggested to support the existence of a unique initiation binding site, topographically distinct from the sites employed during the elongation phase.
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PMID:RNA polymerase. Direct evidence for a unique topographical site for initiation. 390 13

Escherichia coli RNA polymerase was found to bind specifically to restriction fragments containing either end of IS1. DNase I footprint analyses indicate that RNA polymerase protects approximately 70 base-pairs at each end of IS1, including the left or right terminal inverted repeat sequences in IS1 (termed insL or insR, respectively) as well as some non-IS1 sequence directly adjacent to each end of IS1. Analysis of transcripts from the left terminal region of IS1 shows that the insL sequence contains a promoter (named insPL), and that RNA synthesis initiates apparently at one in a stretch of five adenylate residues within insL and continues toward the interior region of IS1. Interestingly, most of the resulting transcripts contain polyuridylate residues (more than 5 U residues) at their 5'-ends. Analysis of transcripts from the right terminal region of IS1 indicates that the insR sequence also contains a promoter (named insPR). RNA synthesis initiates specifically at an adenylate residue within insR and continues toward the interior region of IS1, i.e. in the opposite direction to RNA synthesis initiating at insPL, which is present at the other end of IS1. We propose that insPL is used to make the messenger RNA for the IS1-encoded genes insA and insB, while insPR might be used to synthesize an anti-mRNA and thereby negatively regulate insPL.
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PMID:Both inverted repeat sequences located at the ends of IS1 provide promoter functions. 608 43

The abortive initiation reaction of RNA polymerase has been used to prepare adenylyl-(3'--5')-uridine 5'-phosphate (pApU) in 74% yield from AMP and UTP. The reactive intermediate p-azidophenyl phosphorimidazolidate has been prepared by starting from p-nitrophenyl phosphate. Reaction of this compound with the terminal phosphates of adenosine 5'-phosphate and adenylyl-(3'--5')-uridine 5'-phosphate gives the corresponding beta-substituted 5'-diphosphates. These products are incorporated into the 5' (leading) end of RNA by RNA polymerase (Escherichia coli) and can be photoactivated at a specific stage of RNA elongation. The dinucleotide photoaffinity label beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate stimulates RNA synthesis more strongly than adenylyl-(3'--5')-uridine.
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PMID:Synthesis of mono- and dinucleotide photoaffinity probes of ribonucleic acid polymerase. 616 87

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.
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PMID:Primer specificity of ribosome-associated poly(A) polymerase from Ehrlich ascites tumour cells. 624 52

The various events which result in modification of a primary RNA transcipt may have a role in choosing transcripts which are to be processed into mRNA. If there is a stepwise and interdependent nature to each of the modifications then it is important to place the various steps in temporal order. We show here that the formation of a capped 5' terminus seems to be a very early event for adenovirus type 2 (Ad-2) nuclear RNA that is initiated at the major late Ad-2 promoter. There is an equally rapid entry of 3H-adenosine into the cap and the first dozen or so adenylate residues in the RNA chain. Taken together with evidence on general mRNA metabolism in Chinese hamster cells, it seems unlikely that capping has any differential role in successful RNA processing rather is an automatic event for all RNA polymerase II products.
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PMID:Early capping of transcripts from the adenovirus major late transcription unit. 743 61

Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5'-sulfamoyl (AdoOSO(2)NH(2)) or N-ethylsulfamoyl (AdoOSO(2)NHCH(2)CH(3)) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K(i) values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8-A cocrystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp-123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser-107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.
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PMID:Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors. 1498 31

It has been established that many eukaryotic mRNAs contain poly(adenylic acid) tracts at their 3'-termini. The polyadenylation of mRNA occurs post-transcriptionally in the nucleus as a rapid, initial addition of 100-200 adenylate residues to the pre-mRNA (ref. 1). Subsequently, a slower chain extension (6-8 bases) of the poly(A) tail seems to occur both in the nucleus and in the cytoplasm. The initial polyadenylation reaction can be specifically inhibited by the drug cordycepin (3'-deoxyadenosine) in cell culture, presumably by its conversion to the triphosphate analogue which acts as a competitive inhibitor of poly(A) polymerase. Cordycepin, however, has little effect on the slower poly(A) extension reaction or on the formation of mRNA precursor molecules; but it can inhibit rRNA synthesis. Contrary to the in vitro observations, cordycepin 5'-triphosphate (3'dATP) is not a specific inhibitor of poly(A) synthesis in vivo, relative to RNA synthesis, and RNA polymerase I (which synthesises rRNA) is actually less sensitive to inhibition by 3'dATP than RNA polymerase II (ref. 10) (which is presumed to be involved in the synthesis of mRNA). Since nuclear poly(A) polymerase occurs in two functional states as 'free' and 'chromatin-bound' forms, we reasoned that if the chromatin-associated poly(A) polymerase were involved in the initial polyadenylation of mRNA, it might be selectively inhibited by 3'dATP. The present studies, designed to test such an idea, demonstrate that, as in vivo, the initial polyadenylation reaction can be selectively inhibited in vitro by low levels of 3'dATP. These data also show that higher levels of 3'dATP can inhibit RNA synthesis, 'chromatin-bound' RNA polymerase I activity being significantly more sensitive than the 'bound' RNA polymerase II activity.
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PMID:Specific inhibition of chromatin-associated poly(A) synthesis in vitro by cordycepin 5'-triphosphate. 1607 40

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5'-monophosphate and adenosine 5'-monophosphate (AMP) when it is incubated in the presence of cytidine 5'-triphosphate (CTP), adenosine 5'-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When alpha (32)PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3' end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3' end of which is found in a fragment having a sedimentation constant close to 5s.
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PMID:Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA. 1678 36

Treatment of poly C with methoxyamine leads to formation of 5,6-dihydro-6-methoxyaminocytosine residues in the polymer. These direct incorporation of adenylate residues into the poly G synthesized when the modified poly C is used as a template for RNA polymerase. The process is analogous to that of hydroxylamine-induced errors in replication. Comparison of the uptake of [14C]methoxyamine by the poly C with the percentage of adenylate incorporated in the poly G shows that the induction of errors is a highly efficient process: every modified cytosine residue directs the incorporation of one adenylate residue. A probable explanation for this is that the incorporation is mediated by hydrogen-bonding between the adenine and a predominant imino tautomer of the cytosine adduct.
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PMID:The efficiency of induction of mutations by hydroxylamine. 1976 59

We present an optimized synthetic strategy for the attachment of molecules to 5'-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH>7.5 and releases them at pH<6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.
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PMID:Optimized method for the synthesis and purification of adenosine--folic acid conjugates for use as transcription initiators in the preparation of modified RNA. 2116 52


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