Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase)

Andrei L. Okorokov*, Konstantin I. Panov, Elena Yu Kolbanovskaya, Marat Ya Karpeisky, Konstantin M. Polyakov, Anthony J. Wilkinson, Guy G. Dodson

*Corresponding author for this work

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)143-146
Number of pages4
JournalFEBS Letters
Volume384
Issue number2
DOIs
Publication statusPublished - 15 Apr 1996
Externally publishedYes

Fingerprint

ribonuclease T(2)
Mutagenesis
Ribonucleases
Site-Directed Mutagenesis
Substitution reactions
Amino Acid Substitution
Amino Acids
Poly A
Aptitude
Guanine
Oligonucleotides
Escherichia coli
Hydrolysis
Nucleotides
Kinetics
Substrates

Keywords

  • binase
  • ribonuclease
  • RNase Sa
  • site-directed mutagenesis

Cite this

Okorokov, A. L., Panov, K. I., Kolbanovskaya, E. Y., Karpeisky, M. Y., Polyakov, K. M., Wilkinson, A. J., & Dodson, G. G. (1996). Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase). FEBS Letters, 384(2), 143-146. https://doi.org/10.1016/0014-5793(96)00299-2
Okorokov, Andrei L. ; Panov, Konstantin I. ; Kolbanovskaya, Elena Yu ; Karpeisky, Marat Ya ; Polyakov, Konstantin M. ; Wilkinson, Anthony J. ; Dodson, Guy G. / Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase). In: FEBS Letters. 1996 ; Vol. 384, No. 2. pp. 143-146.
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abstract = "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.",
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Okorokov, AL, Panov, KI, Kolbanovskaya, EY, Karpeisky, MY, Polyakov, KM, Wilkinson, AJ & Dodson, GG 1996, 'Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase)', FEBS Letters, vol. 384, no. 2, pp. 143-146. https://doi.org/10.1016/0014-5793(96)00299-2

Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase). / Okorokov, Andrei L.; Panov, Konstantin I.; Kolbanovskaya, Elena Yu; Karpeisky, Marat Ya; Polyakov, Konstantin M.; Wilkinson, Anthony J.; Dodson, Guy G.

In: FEBS Letters, Vol. 384, No. 2, 15.04.1996, p. 143-146.

Research output: Contribution to journalArticle

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AU - Okorokov, Andrei L.

AU - Panov, Konstantin I.

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AU - Karpeisky, Marat Ya

AU - Polyakov, Konstantin M.

AU - Wilkinson, Anthony J.

AU - Dodson, Guy G.

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