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Vol. 11, No. 21,
pp. 2897-2909,
November 1, 1997
-like factor and core RNA polymerase
1 Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262 USA; 2 Department of Molecular Biology, Taegu University, Taegu, Korea
The cyclic interactions that occur between the subunits of the
yeast mitochondrial RNA polymerase can serve as a simple model for the
more complex enzymes in prokaryotes and the eukaryotic nucleus. We have
used two-hybrid and fusion protein constructs to analyze the
requirements for interaction between the single subunit core polymerase
(Rpo41p), and the 
-like promoter specificity factor (Mtf1p). We were
unable to define any protein truncations that retained the ability to
interact, indicating that multiple regions encompassing the entire
length of the proteins are involved in interactions. We found that 9 of
15 nonfunctional (petite) point mutations in Mtf1p isolated in a
plasmid shuffle strategy had lost the ability to interact. Some of the
noninteracting mutations are temperature-sensitive petite (ts petite);
this phenotype correlates with a precipitous drop in mitochondrial
transcript abundance when cells are shifted to the nonpermissive
temperature. One temperature-sensitive mutant demonstrated a striking
pH dependence for core binding in vitro, consistent with the physical
properties of the amino acid substitution. The noninteracting mutations
fall into three widely spaced clusters of amino acids. Two of the
clusters are in regions with amino acid sequence similarity to
conserved regions 2 and 3 of factors and related proteins; these
regions have been implicated in core binding by both prokaryotic and
eukaryotic -like factors. By modeling the location of the mutations
using the partial structure of Escherichia coli
70, we find that two of the clusters are potentially
juxtaposed in the three-dimensional structure. Our results demonstrate
that interactions between -like specificity factors and core RNA
polymerases require multiple regions from both components of the
holoenzymes.
[Key Words:
RNA polymerase; 
factor; transcription
initiation; mitochondrial transcription; MTF1; RPO41; two-hybrid]
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