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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)
14C-labeled SV40 DNA has been transcribed with E. coli
RNA polymerase
using 3H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4'-
aminomethyl
-4,5', 8-trimethylpsoralen (AMT), at 37 degrees C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) SV40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not required for successful crosslinkage.
...
PMID:Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV40 nascent RNA to its double-stranded DNA template. 20 76
Reovirus infectivity and core-associated
RNA polymerase
activity were decreased by irradiation with long wavelength ultraviolet light in the presence of the 4'-substituted psoralen derivatives, 4'-
aminomethyl
-4,5',8-trimethylpsoralen and 4'-hydroxymethyl-4,5',8-trimethylpsoralen. Monoadduct formation occurred after photoreaction with low psoralen concentrations or brief irradiation times, and the presence of KCl or magnesium acetate had a protective effect. Under the mild reaction conditions in which 1 molecule of 4'-
aminomethyl
-4,5',8-trimethylpsoralen was bound covalently per 160 to 290 base-pairs, the polymerase activity was decreased by greater than 90%. At higher drug concentrations or longer times of photoreaction of reovirus cores, the viral RNA was extensively cross-linked indicating that the reovirus genome in situ is double-stranded.
...
PMID:Photochemical cross-linking of reovirus genome RNA in situ and inactivation of viral transcriptase. 72 5
The interaction of 4'-N(2-aminoethyl)
aminomethyl
-4,5',8-trimethylpsoralen-modified oligonucleoside methylphosphonates with synthetic ds-DNA containing a T7
RNA polymerase
promoter was studied. The oligomers effectively crosslinked with either coding or noncoding ss-DNA when irradiated at 365 nm, but not with ds-DNA. The extent of the crosslinking reaction, which was complete within 16 min: (a) reached its maximum at an oligomer concentration of 3 microM; (b) remained constant below the Tm of the duplex and then rapidly decreased; and (c) appeared to depend upon the sequence surrounding the psoralen crosslinking site. An oligomer crosslinked to the template strand inhibited transcription by T7
RNA polymerase
whereas an oligomer crosslinked to the non-template strand had only a small inhibitory effect. Oligomers did not crosslink to ds-DNA undergoing transcription nor did they inhibit the transcription reaction.
...
PMID:Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with synthetic DNA containing a promoter for T7 RNA polymerase. 306 Aug 47
RNA-RNA interactions between 18S ribosomal RNA and noncapped influenza cRNA were detected by psoralen photochemical cross-linking in reticulocyte 80S ribosome-cRNA complexes. In vitro transcripts of type A influenza virus synthesized by endogenous
RNA polymerase
with adenylyl-(3'----5')-guanosine primer formed 80S complexes with rabbit reticulocyte ribosomes. The extent of the complex formation by these noncapped cRNAs was less than that by the m7G-capped reovirus in vitro transcripts, but the former RNAs in the 80S complexes were cross-linked to 18S rRNA as efficiently as the latter RNAs by photoreaction with an RNA cross-linking agent, 4'-
aminomethyl
-4,5',8-trimethylpsoralen. These results suggested that mRNA with or without the cap structure on the 5'-terminal can form complexes with the ribosomes in a eukaryotic cell-free translation system by base-pair formation with 18S rRNA. Correspondingly, sequences capable of forming extensive base-pairs including four- to five-base complementarities were found between the 3'-terminal of rabbit reticulocyte 18S ribosomal RNA and the 5'-noncoding regions of either influenza virus transcripts or reovirus mRNA.
...
PMID:Base-pair formation between noncapped influenza virus in vitro transcripts and 18S rRNA in rabbit reticulocyte 80S ribosome-mRNA complexes detected by psoralen photoreaction. 670 1
The self-sustained sequence replication (3SR) reaction is an extremely efficient method for amplifying target DNA and RNA sequences that may be present in minute quantities. A serious problem often encountered in its practice is carryover contamination from products of previous 3SR reactions. A postamplification treatment of 3SR reaction products with the photoactive agent 4'-
aminomethyl
-4,5-dimethylisopsoralen (IP-10) was investigated as an approach for preventing carryover contamination by 3SR amplicons. Initially, inhibition of the amplification reaction by high concentrations of the reagent was observed. This problem was circumvented by developing a gel-based delivery of IP-10, and the method was found to provide highly efficient sterilization (approximately 10(6)-fold) of 3SR amplicons. Evaluation of this strategy on a number of 3SR targets has indicated that the degree of sterilization is dependent on the length of the amplified region and on the concentration of IP-10. It appears that the sterilization effect is caused by covalent modification of the pyrimidine bases of RNA and DNA, which renders them unusable as templates for the 3SR reaction. Modification of a purified RNA transcript with IP-10 was shown to prevent effectively reverse transcription by avian myeloblastosis virus reverse transcriptase (AMV RT). Similarly, treatment of a T7
RNA polymerase
promoter-containing DNA template with IP-10 eliminated full-length transcription by T7
RNA polymerase
. This isopsoralen method may be used to sterilize multiple 3SR reactions in a clinical assay with a convenient UV irradiation step.
...
PMID:Photochemical sterilization of 3SR reactions. 750 79
N
6
-methylated adenine (m
6
A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N
6
-methylated AMP (N
6
-mAMP), which has an unclear metabolic fate. We show that
Arabidopsis thaliana
and human cells require an N
6
-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N
6
-mAMP to inosine monophosphate in vivo by hydrolytically removing the
aminomethyl
group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N
6
A-RNA methylation in these organisms. MAPDA likely protects RNA from m
6
A misincorporation. This is required because eukaryotic
RNA polymerase
can use N
6
-mATP as a substrate. Upon abrogation of
MAPDA
, root growth is slightly reduced, and the N
6
-methyladenosine, N
6
-mAMP, and N
6
-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m
6
A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N
6
-mAMP was severalfold more abundant than N
6
-mATP in
MAPDA
mutants, we speculate that additional molecular filters suppress the generation of N
6
-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N
6
-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N
6
-mAMP accumulation and salvage.
...
PMID:m
6
A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N
6
-Methyl-AMP Deaminase in Plant and Mammalian Cells. 2990 27
