Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins.
We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.
Hwang
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YW
Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Sanchez
A
A
Miller
D L
DL
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D012360:RNA, Transfer, Phe / Q000378:metabolism; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D012360:RNA, Transfer, Phe; C023512:elongation factor Ts; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D008239:Lysine