Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor.
The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.
Jana
N K
NK
Department of Biochemistry, Bose Institute, Calcutta, India.
Roy
S
S
Bhattacharyya
B
B
Mandal
N C
NC
D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003062:Codon; D004268:DNA-Binding Proteins; D016296:Mutagenesis; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D003062:Codon; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014364:Tryptophan