Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase).
Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site-directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild-type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate.
Okorokov
A L
AL
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Panov
K I
KI
Kolbanovskaya EYu
Karpeisky MYa
Polyakov
K M
KM
Wilkinson
A J
AJ
Dodson
G G
GG
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000235:genetics*; D006147:Guanine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009843:Oligoribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D006147:Guanine; D004722:Endoribonucleases; C022543:ribonuclease T(2)