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Mol Cell Biol, April 1998, p. 2130-2142, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Functions of the N- and C-Terminal Domains of Human
RAP74 in Transcriptional Initiation, Elongation, and Recycling of
RNA Polymerase II
Lei
Lei,
Delin
Ren,
Ann
Finkelstein, and
Zachary
F.
Burton*
Department of Biochemistry, Michigan State
University, East Lansing, Michigan 48824-1319
Received 4 November 1997/Returned for modification 11 December
1997/Accepted 14 January 1998
Transcription factor IIF (TFIIF) cooperates with RNA polymerase II
(pol II) during multiple stages of the transcription cycle including
preinitiation complex assembly, initiation, elongation, and possibly
termination and recycling. Human TFIIF appears to be an
2
2 heterotetramer of RNA polymerase
II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30). From
inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit
appears to comprise separate N- and C-terminal domains connected by a
flexible loop. In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N-
and C-terminal domains and the central loop of RAP74 have distinct
roles during separate phases of the transcription cycle. The N-terminal
domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II
into a complex formed on the adenovirus major late promoter with the
TATA-binding protein, TFIIB, and RAP30. A more complete N-terminal
domain fragment (aa 1 to 217) strongly stimulates both accurate
initiation and elongation by pol II. The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both
accurate initiation and elongation, and mutations in these regions have
similar effects on initiation and elongation. Based on these
observations, RAP74 appears to have similar functions in initiation and
elongation. The central region and the C-terminal domain of RAP74 do
not contribute strongly to single-round accurate initiation or
elongation stimulation but do stimulate multiple-round transcription in
an extract system.
*
Corresponding author. Mailing address: Department of
Biochemistry, Michigan State University, East Lansing, MI 48824-1319. Phone: (517) 353-0859. Fax: (517) 353-9334. E-mail:
burton{at}pilot.msu.edu.
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