Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.


Abstract

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.coli clones with temperature-sensitive phenotype in the presence of chloramphenicol. The resistance to chloramphenicol used to select these clones results from expression control of the chloramphenicol acetyl transferase gene by the T7 promoter. In a second phase, and using the thermosensitive T7 RNAP variants as template, a new round of random mutagenesis was performed. Combined to an appropriate screening strategy, 11 mutations (second-site T7 RNAP revertants) that restore the initial resistance to chloramphenicol at 42°C were identified. Nine of these mutations increase the thermal resistance of the wild-type T7 RNA. They include the five mutations previously described using different approaches and four novel mutations. One improves T7 RNA catalytic activity and one has no positive effect on the natural enzyme but increases the activity of some combined mutants. Additive effects of mutations amount to an increase of as much as 10°C in T1/2 compared with the wild-type enzyme and up to a 2-fold activity enhancement.

Submission Details

ID: S4URrno63

Submitter: Shu-Ching Ou

Submission Date: Dec. 7, 2018, 1:10 p.m.

Version: 1

Publication Details
Boulain JC;Dassa J;Mesta L;Savatier A;Costa N;Muller BH;L'hostis G;Stura EA;Troesch A;Ducancel F,Protein Eng Des Sel (2013) Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase. PMID:24006372
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1MSW 2002-11-15 2.1 Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase
1QLN 2000-02-04 2.4 STRUCTURE OF A TRANSCRIBING T7 RNA POLYMERASE INITIATION COMPLEX
1CEZ 1999-05-21 2.4 CRYSTAL STRUCTURE OF A T7 RNA POLYMERASE-T7 PROMOTER COMPLEX
2PI4 2007-06-19 2.5 T7RNAP complexed with a phi10 protein and initiating GTPs.
1S77 2004-03-23 2.69 T7 RNAP product pyrophosphate elongation complex
1ARO 1998-10-21 2.8 T7 RNA POLYMERASE COMPLEXED WITH T7 LYSOZYME
1S76 2004-03-23 2.88 T7 RNA polymerase alpha beta methylene ATP elongation complex
1H38 2002-11-20 2.9 Structure of a T7 RNA polymerase elongation complex at 2.9A resolution
2PI5 2007-06-19 2.9 T7 RNA polymerase complexed with a phi10 promoter
4RNP 1997-12-03 3.0 BACTERIOPHAGE T7 RNA POLYMERASE, HIGH SALT CRYSTAL FORM, LOW TEMPERATURE DATA, ALPHA-CARBONS ONLY
3E2E 2008-11-04 3.0 Crystal Structure of an Intermediate Complex of T7 RNAP and 7nt of RNA
1S0V 2004-02-24 3.2 Structural basis for substrate selection by T7 RNA polymerase
3E3J 2008-11-04 6.7 Crystal Structure of an Intermediate Complex of T7 RNAP and 8nt of RNA

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 D T7 RNA polymerase P00573 RPOL_BPT7