Functional coupling of cleavage and polyadenylation with transcription of mRNA

Todd E Adamson; Damon C Shutt; David H Price

doi:10.1074/jbc.M505532200

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Functional coupling of cleavage and polyadenylation with transcription of mRNA

Journal article

Open access

Peer reviewed

Functional coupling of cleavage and polyadenylation with transcription of mRNA

Todd E Adamson, Damon C Shutt and David H Price

The Journal of biological chemistry, Vol.280(37), pp.32262-32271

09/16/2005

DOI: 10.1074/jbc.M505532200

PMID: 16041059

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Abstract

Cleavage and polyadenylation define the 3' ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5' portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5' capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.

Protein Structure, Tertiary

Humans

Poly A - chemistry

Positive Transcriptional Elongation Factor B - metabolism

RNA - chemistry

RNA, Messenger - metabolism

Blotting, Western

Chymotrypsin - pharmacology

DNA - chemistry

Salts - pharmacology

Cell Nucleus - metabolism

Macromolecular Substances - chemistry

Time Factors

Polyadenylation

Polymerase Chain Reaction

Chymotrypsin - chemistry

Transcription, Genetic

HeLa Cells

Adenosine Triphosphate - chemistry

Potassium Chloride - chemistry

DNA, Complementary - metabolism

RNA Polymerase II - chemistry

Details

Title: Subtitle: Functional coupling of cleavage and polyadenylation with transcription of mRNA
Creators: Todd E Adamson - Department of Biochemistry, University of Iowa, Iowa City, 52242, USA
Damon C Shutt
David H Price
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.280(37), pp.32262-32271
DOI: 10.1074/jbc.M505532200
PMID: 16041059
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM35500 / NIGMS NIH HHS
Language: English
Date published: 09/16/2005
Academic Unit: Nursing; Biochemistry and Molecular Biology
Record Identifier: 9984024554302771

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