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.
Amber mutations have been constructed at 328 positions, corresponding to residues 2 to 329 in the E. coli lac repressor protein. Synthetic and naturally occurring nonsense suppressors have been used to insert, in series, 12-13 amino acids at positions specified by an amber (UAG) codon in the lacI mRNA. The resulting set of over 4000 single amino acid replacements in the lac repressor protein allows a detailed analysis of its substitution tolerance along the linear array of residues, and reveals structure-function relationships in lac repressor and in proteins in general. (1) There are two main regions in the repressor which are extremely sensitive to amino acid replacements. One, the amino-terminal 59 residues, has been implicated in DNA and operator binding by a large body of work. The second, extending from approximately residues 239 to 289/292, forms the repressor core and shares the most homology with other repressor and DNA binding proteins. (2) Throughout the rest of the protein, segments of 6 to 14 amino acids, which are highly tolerant to single amino acid replacements, appear to act as "spacers" between one or several hydrophobic residues that are relatively intolerant to substitutions. (3) We have replaced the amino acids in these tolerant regions with spans of alanine residues, from 5 to 13 amino acids. In all five of the regions tested, alanine replacements, sometimes of up to 8 amino acids, still allowed functional repressor, while deletion of the same residues destroyed repressor function. This reinforces the view that many regions of a protein do not require a specific sequence to serve as spacers between more important residues. (4) A distinct pattern of substitutions leading to the I(s) phenotype suggests the location of residues involved in inducer binding. (5) A number of general substitution patterns can be recognized. For instance, proline is not tolerated at over 40 sites which tolerate all the other amino acid replacements. Another set of sites tolerates only non-polar amino acids, whereas a third set tolerates a subset of the smallest amino acids, (serine, alanine, glycine and cysteine, and sometimes threonine and valine). (5) Overall, 93 of 328 sites (28%) tolerate all 13 amino acids tested, and 144 of 328 (44%) tolerate 12/13 or all 13 substitutions. We judge that 192 of 328 sites (59%) are generally tolerant to substitutions.
Markiewicz
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P
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Kleina
L G
LG
Cruz
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C
Ehret
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S
Miller
J H
JH
D000409:Alanine / Q000502:physiology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D056968:Lac Repressors; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D010641:Phenotype; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D001616:beta-Galactosidase; D000409:Alanine