Protein and DNA requirements for the transcription factor IIIA-induced distortion of the 5 S rRNA gene promoter

被引:11
作者
Brown, ML
Schroth, GP
Gottesfeld, JM
BazettJones, DP
机构
[1] UNIV CALGARY, DEPT BIOCHEM MED, CALGARY, AB T2N 4N1, CANADA
[2] OREGON STATE UNIV, DEPT BIOCHEM & BIOPHYS, CORVALLIS, OR 97331 USA
[3] Scripps Res Inst, DEPT MOL BIOL, LA JOLLA, CA 92037 USA
关键词
DNA-protein complexes; transcription factor IIIA; DNA-bending proteins; electron spectroscopic imaging; zinc fingers;
D O I
10.1006/jmbi.1996.0539
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
Transcription factor-induced DNA distortion has become a common theme in eukaryotic gene regulation. A number of techniques have been applied to the study of transcription factor-induced DNA bending and flexibility including electron microscopy, circular permutation gel analysis, helical phasing gel analysis and cyclisation kinetics in solution. We have applied these techniques in order to assess the role that specific DNA sequences and protein domains of transcription factor IIIA (TFIIIA) play in the TFIIIA-induced distortion of the Xenopus 5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA:DNA complexes indicate that TFIIIA binding involves compaction of the 5 S promoter into a precise three-dimensional hairpin-shaped structure. This compaction can be detected utilising circular permutation gel analysis and the distortion results in an apparent bend angle of 55 to 60 degrees near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60 degrees bend angle as measured by circular permutation can be detected as a static bend directed towards the minor groove between bases +63 and +64 of the 5 S rRNA gene, The amplitude of the TFIIIA:5 S gene phasing signal is similar to the phasing signal obtained utilising bacterial CAP:DNA complexes with bend angles of approximately 90 degrees. These results are supported by phased ligase-mediated cyclisation kinetics in solution. Analysis of DNA deletion constructs indicate that the 5' A block of the internal 5 S gene promoter, which is required for transcriptional activity, is also required for TFIIIA-induced distortion of the 55 gene promoter. Analysis of the N-terminal papain fragment of TFIIIA indicates that the 34 kDa zinc finger DNA binding domain is sufficient for compaction of the 5 S gene promoter. These results are discussed in relation to the modular model of TFIIIA:DNA interaction in which individual zinc fingers contribute to the protein-induced distortion of the DNA helix and overall DNA binding affinity in a complex, non-additive fashion. (C) 1996 Academic Press Limited
引用
收藏
页码:600 / 614
页数:15
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