Genetic studies of the lac repressor. XIV: Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as spacers which do not require a specific sequence

摘要: Amber mutations have been constructed at 328 positions, corresponding to residues 2 329 in the E. coli lac repressor protein. Synthetic and naturally occurring nonsense suppressors used insert, series, 12-13 amino acids positions specified by an amber (UAG) codon lacI mRNA. The resulting set of over 4000 single acid replacements protein allows a detailed analysis its substitution tolerance along linear array residues, reveals structure-function relationships proteins general. (1) There are two main regions which extremely sensitive replacements. One, amino-terminal 59 has implicated DNA operator binding large body work. second, extending from approximately 239 289/292, forms core shares most homology with other proteins. (2) Throughout rest protein, segments 6 14 acids, highly tolerant replacements, appear act as "spacers" between one or several hydrophobic that relatively intolerant substitutions. (3) We replaced these spans alanine 5 13 acids. In all five tested, sometimes up 8 still allowed functional repressor, while deletion same destroyed function. This reinforces view many do not require specific sequence serve spacers more important residues. (4) A distinct pattern substitutions leading I(s) phenotype suggests location involved inducer binding. (5) number general patterns can be recognized. For instance, proline is tolerated 40 sites tolerate Another tolerates only non-polar whereas third subset smallest (serine, alanine, glycine cysteine, threonine valine). Overall, 93 (28%) 144 (44%) 12/13 judge 192 (59%) generally