Nucleic Acids Research, 1990, Vol. 18, No. 15 4571-4578
© 1990
MOLECULAR BIOLOGY |
On the flexible interaction of yeast factor r with the bipartite promoter of tRNA genes
Departement de Biologie, Service de Biochimie, Centre d'Etudes Nucleates de Saclay 91191 Gifsur-Yvette Cedex, France 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School 55 Lake Avenue North, Worcester, MA 01655, USA
*To whom correspondence should be addressed
Received February 22, 1990. Revised June 26, 1990. Accepted June 26, 1990.
Yeast transcription factor r (analogous to vertebrate TFIIIC) interacts specifically with the internal split promoter of tRNA genes. Binding to the two promoter elements (A block and B block) occurs within 30 seconds even when they are separated by a long intervening sequence. Dimethylsulfate protection analysis of contact points between r and the noncoding strand of a series of internally deleted tRNALeu3 genes shows that the specificity of the interaction is not affected by changes in the distance or in the relative helical orientation of the promoter elements. This result is consistent with the results of previous footprinting experiments (Baker, R.E., Camier, S., Sentenac, A. and Hall, B.D., 1987, Proc. Nat I. Acad. Sci. USA, 84,8768-8772). To test if any physical constraint is imposed on the DNA molecule upon r binding, we analyzed the effect of introducing random single-strand breaks in the noncoding strand of the tRNA gene. Whereas some nicks located in the A block were found to prevent r binding, no single-strand break in the B block region or in the DNA between the A and B blocks were observed to inhibit or facilitate the binding of r. We therefore propose that the great flexibility of the T-tDNA interaction is mostly due to the T protein itself.
+Present address: Department of Biochemistry, SJ-70, University of Washington, Seattle, WA 98195, USA