scholarly journals Oxazinomycin arrests RNA polymerase at the polythymidine sequences

2019 ◽  
Vol 47 (19) ◽  
pp. 10296-10312 ◽  
Author(s):  
Ranjit K Prajapati ◽  
Petja Rosenqvist ◽  
Kaisa Palmu ◽  
Janne J Mäkinen ◽  
Anssi M Malinen ◽  
...  
Keyword(s):  
Oxygen Atom ◽  
Rna Polymerase ◽  
Inhibitory Effect ◽  
Trojan Horse ◽  
Rna Polymerases ◽  
Streptomyces Hygroscopicus ◽  
Rna Cleavage ◽  
Sequence Context ◽  
Good Substrate ◽  
Dna Template

Abstract Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

scholarly journals Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

2021 ◽  
Author(s):  
Akihito Fukudome ◽  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Eswar Reddem ◽  
Francisco Martinez-Marquez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Rna Polymerases ◽  
Rna Recognition Motif ◽  
Rna Dependent Rna Polymerase ◽  
Guide Rna ◽  
Pol Iv ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Template

AbstractRNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3’ ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNA’s 3’ end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3’ end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3’ end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand.SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3’ end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

RNAP subunits F/E (RPB4/7) are stably associated with archaeal RNA polymerase: using fluorescence anisotropy to monitor RNAP assembly in vitro

2009 ◽  
Vol 421 (3) ◽  
pp. 339-343 ◽  
Author(s):  
Dina Grohmann ◽  
Angela Hirtreiter ◽  
Finn Werner
Keyword(s):  
Rna Polymerase ◽  
Molecular Interactions ◽  
Fluorescence Anisotropy ◽  
Rna Polymerases ◽  
Dynamic Exchange ◽  
Transcription Cycle

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

The REB1 site is an essential component of a terminator for RNA polymerase I in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 649-658
Author(s):  
W H Lang ◽  
R H Reeder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Protein Binding ◽  
Binding Site ◽  
Rna Polymerase I ◽  
Recognition Sequence ◽  
Sequence Context ◽  
Yeast Saccharomyces Cerevisiae ◽  
Essential Component ◽  
Polymerase I

We have identified a terminator for transcription by RNA polymerase I in the genes coding for rRNA of the yeast Saccharomyces cerevisiae. The terminator is located 108 bp downstream of the 3' end of the mature 25S rRNA and shares several characteristics with previously studied polymerase I terminators in the vertebrates. For example, the yeast terminator is orientation dependent, is inhibited by its own sequence, and forms RNA 3' ends 17 +/- 2 bp upstream of an essential protein binding site. The recognition sequence for binding of the previously cloned REB1 protein (Q. Ju, B. E. Morrow, and J. R. Warner, Mol. Cell. Biol. 10:5226-5234, 1990) is an essential component of the terminator. In addition, the efficiency of termination depends upon sequence context extending at least 12 bp upstream of the REB1 site.

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