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Molecular and Cellular Biology, November 1999, p. 7377-7387, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Region within the RAP74 Subunit of Human
Transcription Factor IIF Is Critical for Initiation but Dispensable
for Complex Assembly
Delin
Ren,
Lei
Lei, and
Zachary F.
Burton*
Department of Biochemistry, Michigan State
University, East Lansing, Michigan 48824-1319
Received 19 April 1999/Returned for modification 24 May
1999/Accepted 3 August 1999
Human transcription factor IIF (TFIIF) is an
2
2 heterotetramer of RNA polymerase
II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows
that the N-terminal region of RAP74 between L155 (leucine at codon 155)
and M177 is important for initiation. Mutants in this region have
reduced activity in transcription, but none are inactive. Single amino
acid substitutions at hydrophobic residues L155, W164, I176, and M177
have similar activity to RAP74(1-158), from which all but three amino
acids of this region are deleted. Residual activity can be explained
because each of these mutants forms a complex with RAP30 and recruits
RNA polymerase II into the preinitiation complex. Mutants are defective
for formation of the first phosphodiester bond from the adenovirus
major late promoter but do not appear to have an additional significant
defect in promoter escape. Negative DNA supercoiling partially
compensates for the defects of TFIIF mutants in initiation, indicating
that TFIIF may help to untwist the DNA helix for initiation.
*
Corresponding author. Mailing address: Department of
Biochemistry, Michigan State University, E. Lansing, MI 48824-1319. Phone: (517) 353-0859. Fax: (517) 353-9334. E-mail:
burton{at}pilot.msu.edu.
Molecular and Cellular Biology, November 1999, p. 7377-7387, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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