THE HIP1 INITIATOR ELEMENT PLAYS A ROLE IN DETERMINING THE INVITRO REQUIREMENT OF THE DIHYDROFOLATE-REDUCTASE GENE PROMOTER FOR THE C-TERMINAL DOMAIN OF RNA POLYMERASE-II

被引:50
作者
BUERMEYER, AB [1 ]
THOMPSON, NE [1 ]
STRASHEIM, LA [1 ]
BURGESS, RR [1 ]
FARNHAM, PJ [1 ]
机构
[1] UNIV WISCONSIN,MCARDLE LAB CANC RES,MADISON,WI 53706
关键词
D O I
10.1128/MCB.12.5.2250
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.
引用
收藏
页码:2250 / 2259
页数:10
相关论文
共 51 条
[1]   THE C-TERMINAL DOMAIN OF THE LARGEST SUBUNIT OF RNA POLYMERASE-II OF SACCHAROMYCES-CEREVISIAE, DROSOPHILA-MELANOGASTER, AND MAMMALS - A CONSERVED STRUCTURE WITH AN ESSENTIAL FUNCTION [J].
ALLISON, LA ;
WONG, JKC ;
FITZPATRICK, VD ;
MOYLE, M ;
INGLES, CJ .
MOLECULAR AND CELLULAR BIOLOGY, 1988, 8 (01) :321-329
[2]   MUTATIONS IN RNA POLYMERASE-II ENHANCE OR SUPPRESS MUTATIONS IN GAL4 [J].
ALLISON, LA ;
INGLES, CJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (08) :2794-2798
[3]  
ARIAS JA, 1991, J BIOL CHEM, V266, P8055
[4]   GENETIC-ANALYSIS OF THE REPETITIVE CARBOXYL-TERMINAL DOMAIN OF THE LARGEST SUBUNIT OF MOUSE RNA POLYMERASE-II [J].
BARTOLOMEI, MS ;
HALDEN, NF ;
CULLEN, CR ;
CORDEN, JL .
MOLECULAR AND CELLULAR BIOLOGY, 1988, 8 (01) :330-339
[5]   MOLECULAR-ORGANIZATION OF THE HUMAN RAF-1 PROMOTER REGION [J].
BECK, TW ;
BRENNSCHEIDT, U ;
SITHANANDAM, G ;
CLEVELAND, J ;
RAPP, UR .
MOLECULAR AND CELLULAR BIOLOGY, 1990, 10 (07) :3325-3333
[6]  
BORELLI MJ, 1987, EXP CELL RES, V170, P363
[7]  
BUERMEYER AB, UNPUB
[8]   TRANSCRIPTION INITIATION-COMPLEXES AND UPSTREAM ACTIVATION WITH RNA POLYMERASE-II LACKING THE C-TERMINAL DOMAIN OF THE LARGEST SUBUNIT [J].
BURATOWSKI, S ;
SHARP, PA .
MOLECULAR AND CELLULAR BIOLOGY, 1990, 10 (10) :5562-5564
[9]   PHOSPHORYLATION OF RNA-POLYMERASE BY THE MURINE HOMOLOG OF THE CELL-CYCLE CONTROL PROTEIN-CDC2 [J].
CISEK, LJ ;
CORDEN, JL .
NATURE, 1989, 339 (6227) :679-684
[10]   TAILS OF RNA POLYMERASE-II [J].
CORDEN, JL .
TRENDS IN BIOCHEMICAL SCIENCES, 1990, 15 (10) :383-387