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Volume 270, Number 10, Issue of March 10, 1995 pp. 5107-5114
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis of the COOH-terminal Region of Bacteriophage T4 regA Protein (*)

(Received for publication, July 14, 1994; and in revised form, December 18, 1994)

Shawn M. O'Malley (1),  A. K. M. Sattar (1),  Kenneth R. Williams (2),  Eleanor K. Spicer (3)(§)

From the  (1)Department of Molecular Biophysics and Biochemistry and (2)Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 and (3)Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT

The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of >12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT) occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the K of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 194 and 1109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.

FOOTNOTES

*
This work was primarily supported by National Science Foundation Grants MCB-9118617 and MCB-9496143 (to E. K. S.), with limited support also from National Institutes of Health Grant GM31539 (to K. R. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-4321; Fax: 803-792-4322.

(^1)
The abbreviations used are: WT, wild type; PAGE, polyacrylamide gel electrophoresis; LDMS, laser desorption mass spectrometry; RNP, ribonucleoprotein.

(^2)
C.-H. Kang, R. Chan, I. Berger, C. Lockshin, L. Green, L. Gold, and A. Rich(1995), manuscript submitted for publication.

ACKNOWLEDGEMENTS

We are grateful to William Konigsberg for his continuing interest in this work. We thank John Lee for assistance with purification of truncation mutants, Yvonne Bernie for purification of F106V regA protein, and Dr. Lynn Reagen for use of the circular dichroism spectropolarimeter. We gratefully acknowledge the skillful DNA sequencing, protein chemistry, and LDMS analyses performed by the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University). We also thank Ann Ewing and Burnett G. Bryant for careful preparation of this manuscript.


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