Site-directed mutants of post-translationally modified sites of yeast eEF1A using a shuttle vector containing a chromogenic switch.
Eukaryotic elongation factor 1A (eEF1A, formerly eEF-1 alpha) carries aminoacyl-tRNAs into the A-site of the ribosome in a GTP-dependent manner. In order to probe the structure/function relationships of eEF1A, we have generated site-directed mutants using a modification of a highly versatile yeast shuttle vector, which consists of the insertion of a 66 base long synthetic DNA fragment in the vector's polylinker. Via oligonucleotide-directed mutagenesis, the modification permits the identification of mutant clones based on a chromogenic screen of beta-galactosidase activity. Mutagenesis reactions are performed with two or more oligonucleotides, one introducing the chromogenic shift, and the other(s) introducing the mutation(s) of interest in eEF1A. Several rounds of chromogenic shifts and additional mutations can be performed in succession on the same vector. To address the possible function of the methylated lysines in yeast eEF1A, we have changed the post-translationally modified lysines (residue 30, 79, 316 and 390) to arginines using the above methodology. Yeast with eEF1A mutants that substitute arginine in all four sites do not show any phenotypic change. There is also an apparent equivalency of wild-type and mutant yeast eEF1A in in vitro assays. It is concluded that the post-translational modifications of eEF1A are not of major importance for eEF1A's role in translation.
Cavallius
J
J
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935, USA. jxc15@po.cwru.edu
Popkie
A P
AP
Merrick
W C
WC
D005656:Fungal Proteins / Q000096:biosynthesis; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000502:physiology; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides; D010641:Phenotype; D014176:Protein Biosynthesis / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D001616:beta-Galactosidase / Q000235:genetics
D005656:Fungal Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; C012001:polyphenylalanine; D001616:beta-Galactosidase; D008239:Lysine