Multiple Regions on the Escherichia coliHeat Shock Transcription Factor ς32 Determine Core RNA Polymerase Binding Specificity

ABSTRACT We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoHgene, which encodes ς32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimatedKs s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς32 and that differences in contact sites probably exist among sigma factors.

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tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3596-3603.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3596-3603

Author(s):

H M Dunstan ◽

L S Young ◽

K U Sprague

Keyword(s):

Rna Polymerase ◽

Dna Cleavage ◽

Rna Polymerase Iii ◽

Low Frequency ◽

In Vitro Transcription ◽

Class Iii ◽

Transcriptional Inhibition ◽

Transcription In Vitro

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

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Faculty Opinions recommendation of Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1087809.540736 ◽

2007 ◽

Author(s):

Jim Maher

Keyword(s):

Rna Polymerase ◽

De Novo ◽

In Vitro Transcription ◽

E Coli ◽

6S Rna ◽

Binding Inhibition ◽

Polymerase Binding

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tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3596 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3596-3603 ◽

Cited By ~ 3

Author(s):

H M Dunstan ◽

L S Young ◽

K U Sprague

Keyword(s):

Rna Polymerase ◽

Dna Cleavage ◽

Rna Polymerase Iii ◽

Low Frequency ◽

In Vitro Transcription ◽

Class Iii ◽

Transcriptional Inhibition ◽

Transcription In Vitro

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The RNA Polymerase of Influenza Virus, Bound to the 5′ End of Virion RNA, Acts in cis To Polyadenylate mRNA

Journal of Virology ◽

10.1128/jvi.72.10.8214-8219.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8214-8219 ◽

Cited By ~ 40

Author(s):

Leo L. M. Poon ◽

David C. Pritlove ◽

Jane Sharps ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Point Mutations ◽

Conserved Sequence ◽

Reverse Transcription Pcr ◽

Sequence Elements ◽

Complete Set ◽

Polymerase Binding ◽

In Cis

ABSTRACT We previously demonstrated, by limited mutagenesis, that conserved sequence elements within the 5′ end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further characterize the nucleotide residues at the 5′ end of vRNA which might be involved in polyadenylation, a complete set of short and long model vRNA-like templates with mutations at nucleotides 1′ to 13′ (prime notation denotes numbering from the 5′ end) of vRNA were synthesized and transcribed in vitro. The products were assayed for mRNA production with both reverse transcription-PCR and [α-32P]ATP incorporation assays. Results from these independent assays showed that vRNA templates with point mutations at positions 2′, 3′, 7′ to 9′, and 11′ to 13′ synthesized polyadenylated transcripts inefficiently compared with those with mutations at positions 1′, 4′ to 6′, and 10′. Positions 2′, 3′, 7′ to 9′, and 11′ are known to be involved in RNA polymerase binding. Furthermore, residues at positions 11′ to 13′ are known to be involved in base pairing between the 3′ and 5′ ends of vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5′ end of the template vRNA, which must then interact with the 3′ end of the same template for polyadenylation to occur. These results support a model in which acis-acting RNA polymerase is required for the polyadenylation of influenza virus.

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