Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase

Bernard Y. Amegadzie, Byung-Yoon Ahn, Bernard Moss

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized. An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome. Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes. One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation. The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit. Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions. RNA analyses indicated that the 5′-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence.

Original languageEnglish
Pages (from-to)13712-13718
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number21
Publication statusPublished - 1991 Dec 1
Externally publishedYes

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Gene encoding
Vaccinia virus
DNA-Directed RNA Polymerases
Viruses
Gene Expression
Peptides
Open Reading Frames
RNA
Messenger RNA
Proteins
Viral Genome
Reticulocytes
Consensus Sequence
Virus Diseases
Eukaryota
DNA Sequence Analysis
Immunoprecipitation
Recombinant Proteins
Base Pairing
Virion

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase. / Amegadzie, Bernard Y.; Ahn, Byung-Yoon; Moss, Bernard.

In: Journal of Biological Chemistry, Vol. 266, No. 21, 01.12.1991, p. 13712-13718.

Research output: Contribution to journalArticle

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