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J. Biol. Chem., Vol. 276, Issue 34, 31891-31896, August 24, 2001
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70 in the
Nucleation and Maintenance of the Single-stranded DNA Bubble in Open
RNA Polymerase-Promoter Complexes*
,,
From the Department of Biochemistry, Case Western Reserve
University, Cleveland, Ohio 44106-4935
Amino acid residues in region 2 of
70 have been shown to play an important role in
the strand separation step that is necessary for formation of the
functional or open RNA polymerase-promoter complex. Here we present a
comparison of the roles of basic and aromatic amino acids in the
accomplishment of this process, using RNA polymerase bearing alanine
substitutions for both types of amino acids in region 2. We determined
the effects of the substitutions on the kinetics of open complex
formation, as well as on the ability of the RNA polymerase to form
complexes with single-stranded DNA, and with forked DNA duplexes
carrying a single-stranded overhang consisting of bases in the 
10
region. We concluded that two basic amino acids
(Lys414 and Lys418) are important for
promoter binding and demonstrated distinct roles, at a subsequent step,
for two aromatic amino acids (Tyr430 and
Trp433). It is likely that these four amino acids, which
are close to each other in the structure of 70, together
are involved in the nucleation of the strand separation process.
The first two authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Biochemistry,
School of Medicine, Case Western Reserve University, 10900 Euclid Ave.,
Cleveland, OH 44106-4935; Tel.: 216-368-3684; Fax: 216-368-4544;
E-mail: pld2@po.cwru.edu.
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