The measurement of upper-air density and temperature by two radar-tracked falling spheres : technical reportInteractions of Saccharomyces cerevisiae transcription factors with promoters for RNA polymerases II and III.

Abstract

Initiation of transcription by an RNA polymerase is a result of complex signal systems in which proteins and DNA sequences interact to direct transcription. This thesis project involved the study of two proteins from Saccharomyces cerevisiae, TFIIIC and ACE1, that are involved in the initiation of transcription by RNA polymerases III and II. tRNA genes are transcribed by RNA polymerase III. This thesis work determined optimal conditions for the in vitro transcription of tRNA genes by a yeast extract, and this in vitro transcription assay was then used to follow the separation of RNA pol III transcription components by standard ion exchange chromatography techniques. One component, TFIIIC, was shown to bind to the internal promoter sequences of tRNA genes. TFIIIC was further purified on a DNA affinity column. A gel mobility shift assay was developed and was used to show that TFIIIC does not contain phosphate residues that are accessible to acid or alkaline phosphatase, nor does it contain GlcNac, glucosyl, or mannosyl residues. TFIIIC binds to the internal promoters of tRNA genes, the A and B boxes, in which the A-B box distance varies from 30-93 base pairs. This binding is not affected by the relative helical orientation of the promoters. A model that accounts for this flexibility in the location of the binding sites is one in which the binding of TFIIIC to both the A and B boxes causes the DNA in between to bend. A circular permutation gel electrophoresis assay was used to test this model. The results indicated that the binding of TFIIIC to various tRNA genes did not induce a significant bend. TFIIIC-tRNA gene complexes were also treated with KMnO$\sb4$, which preferentially reacts with single-stranded DNA. This data showed that TFIIIC binding did not induce any detectable regions of single-stranded DNA. Thus, no evidence was obtained for either bending or melting of the DNA helix upon binding of TFIIIC to tRNA genes. Saccharomyces cerevisiae responds to elevated environmental metal concentrations by activating transcription of the metallothionein gene (CUP1) by RNA polymerase II. The unlinked ACE1 gene is essential for copper-inducible transcription of CUP1. In this work, a trpE-ACE1 fusion protein expressed in E. coli was shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. DNA binding experiments using target sequences containing only one or two of the binding regions suggested no obligatory cooperativity for binding. These results indicate that the copper-activated ACE1 protein interacts with multiple distinct regulatory regions to efficiently activate CUP1 transcription.