Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins.

We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.

[1]  M Karplus,et al.  Solution NMR studies of intact lambda repressor. , 1983, Journal of biomolecular structure & dynamics.

[2]  C Yanofsky,et al.  Tryptophan biosynthesis in Escherichia coli. Genetic determination of the proteins involved. , 1971, JAMA.

[3]  J Messing,et al.  A system for shotgun DNA sequencing. , 1981, Nucleic acids research.

[4]  M. Ptashne,et al.  Repressor structure and the mechanism of positive control , 1983, Cell.

[5]  L. Reichardt.  Control of bacteriophage lambda repressor synthesis: regulation of the maintenance pathway of the cro and cI products. , 1975, Journal of molecular biology.

[6]  E. Cox.  Bacterial mutator genes and the control of spontaneous mutation. , 1976, Annual review of genetics.

[7]  H. Scheraga.  Use of random copolymers to determine the helix-coil stability constants of the naturally occurring amino acids , 1978 .

[8]  B. Matthews,et al.  Structure of the cro repressor from bacteriophage λ and its interaction with DNA , 1981, Nature.

[9]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[10]  A. Jeffrey,et al.  How the λ repressor and cro work , 1980, Cell.

[11]  F. Blattner,et al.  Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance. , 1974, Virology.

[12]  Mark Ptashne,et al.  Regulatory functions of the λ repressor reside in the amino-terminal domain , 1979, Nature.

[13]  C. Pabo,et al.  The operator-binding domain of λ repressor: structure and DNA recognition , 1982, Nature.

[14]  Stanley Maloy,et al.  Selection for loss of tetracycline resistance by Escherichia coli , 1981, Journal of bacteriology.

[15]  M Ptashne,et al.  Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. , 1980, Journal of molecular biology.

[16]  W. Sly,et al.  Mechanism of λc17cI virulence , 1969 .

[17]  M. Ptashne,et al.  Maximizing gene expression on a plasmid using recombination in vitro , 1978, Cell.

[18]  R. Sauer,et al.  Protein-DNA recognition. , 1984, Annual review of biochemistry.

[19]  B. Weiss,et al.  Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli , 1982, Journal of bacteriology.

[20]  Thomas A. Steitz,et al.  Structure of catabolite gene activator protein at 2.9 Å resolution suggests binding to left-handed B-DNA , 1981, Nature.