Genetic analysis of Xenopus transcription factor IIIA

被引:7
作者
Bumbulis, MJ
Wroblewski, G
McKean, D
Setzer, DR [1 ]
机构
[1] Case Western Reserve Univ, Sch Med, Dept Mol Biol & Microbiol, Cleveland, OH 44106 USA
[2] Baldwin Wallace Coll, Dept Biol, Berea, OH 44017 USA
关键词
TFIIIA; zinc finger; 5 S RNA; DNA-binding; transcription;
D O I
10.1006/jmbi.1998.2285
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 describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TF;IIIA). In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences. This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity. We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity. Over 30 such mutants have been identified and partially characterized. The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene. Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available. Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast. (C) 1998 Academic Press.
引用
收藏
页码:1307 / 1322
页数:16
相关论文
共 59 条
[1]  
ARCHAMBAULT J, 1992, J BIOL CHEM, V267, P3282
[3]   EXPRESSION OF HETEROLOGOUS GENES IN SACCHAROMYCES-CEREVISIAE FROM VECTORS UTILIZING THE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE GENE PROMOTER [J].
BITTER, GA ;
EGAN, KM .
GENE, 1984, 32 (03) :263-274
[4]  
BROW DA, 1987, J BIOL CHEM, V262, P13953
[5]   ELECTRON-MICROSCOPIC STUDY OF CRYSTALS OF THE XENOPUS-LAEVIS TRANSCRIPTION FACTOR-IIIA-5S RIBOSOMAL-RNA COMPLEX [J].
BROWN, RS ;
FERGUSON, C ;
KINGSWELL, A ;
WINKLER, FK ;
LEONARD, KR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (11) :3802-3804
[6]  
BUMBULIS MJ, 1997, NOVEL GENETIC SCREEN
[7]   A MECHANISM FOR SYNERGISTIC ACTIVATION OF A MAMMALIAN GENE BY GAL4 DERIVATIVES [J].
CAREY, M ;
LIN, YS ;
GREEN, MR ;
PTASHNE, M .
NATURE, 1990, 345 (6273) :361-364
[8]   A ROLE IN DNA-BINDING FOR THE LINKER SEQUENCES OF THE 1ST 3 ZINC FINGERS OF TFIIIA [J].
CHOO, Y ;
KLUG, A .
NUCLEIC ACIDS RESEARCH, 1993, 21 (15) :3341-3346
[9]   SEQUENCE-SPECIFIC BINDING OF THE N-TERMINAL 3-FINGER FRAGMENT OF XENOPUS TRANSCRIPTION FACTOR-IIIA TO THE INTERNAL CONTROL REGION OF A 5S RNA GENE [J].
CHRISTENSEN, JH ;
HANSEN, PK ;
LILLELUND, O ;
THOGERSEN, HC .
FEBS LETTERS, 1991, 281 (1-2) :181-184
[10]   DEFINITION OF THE BINDING-SITES OF INDIVIDUAL ZINC FINGERS IN THE TRANSCRIPTION FACTOR-IIIA-5S RNA GENE-COMPLEX [J].
CLEMENS, KR ;
LIAO, XB ;
WOLF, V ;
WRIGHT, PE ;
GOTTESFELD, JM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (22) :10822-10826