Systematic site-directed mutagenesis of protein SRP19. Identification of the residues essential for binding to signal recognition particle RNA.
Protein SRP19 is an important structural and functional constituent of the signal recognition particle (SRP) and belongs to a group of RNA binding proteins that specifically recognize certain tetranucleotide loops. Systematic site-directed mutagenesis was used to identify the amino acid residues in human SRP19 essential for interaction with SRP RNA. In our studies, three different groups of mutants were constructed, and each group covered essentially the entire sequence of the SRP19 protein in a consecutive nonoverlapping fashion. Results from 10 deletion mutants followed by the analysis of 24 mutants in which adjacent five residues were changed to pentaglycine suggested that a large portion of the protein may be required for RNA binding. Further examination of 53 mutant polypeptides in which adjacent dipeptide segments were altered showed, however, that 84 of the 144 SRP19 amino acids (58%) were not important for binding to the SRP RNA. The essential amino acids cluster in five regions which encompass most of the SRP19 sequence, with the exception of residues located at the N and C termini and a predicted internal loop. The results from the systematic site-directed mutagenesis study, when combined with protein secondary structure calculations, demonstrate that SRP19 is a precisely tooled protein which associates intimately with the SRP RNA.
Chittenden
K
K
Department of Molecular Biology, University of Texas Health Science Center, Tyler 75710.
Black
S D
SD
Zwieb
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D017384:Sequence Deletion; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D018271:Signal Recognition Particle; D012313:RNA