A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions.


Abstract

A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described. E. coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot. Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability. Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E. coli RNase HI. Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available. Study holds ProTherm entries: 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 13382, 13383, 13384, 13385, 13386, 13387, 13388, 13389, 13390, 13391, 13392, 13393, 13394, 13395, 13396, 13397, 13398, 13399, 13400, 13401, 13402, 13403, 13404, 13405, 13406, 13407, 13408, 13409, 13410, 13411 Extra Details: Activity determined at 30 degree C Escherichia coli ribonuclease HI; thermostability ;,mutagenesis; protein stability

Submission Details

ID: TehmbBek

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Haruki M;Noguchi E;Akasako A;Oobatake M;Itaya M;Kanaya S,J. Biol. Chem. (1994) A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. PMID:7929430
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
4Z0U 2015-03-26T00:00:00+0000 2.0 RNase HI/SSB-Ct complex
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1F21 2000-05-22T00:00:00+0000 1.4 DIVALENT METAL COFACTOR BINDING IN THE KINETIC FOLDING TRAJECTORY OF E. COLI RIBONUCLEASE HI
1G15 2000-10-10T00:00:00+0000 1.9 CO-CRYSTAL OF E. COLI RNASE HI WITH TWO MN2+ IONS BOUND IN THE THE ACTIVE SITE
1GOA 1993-05-10T00:00:00+0000 1.9 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
1GOB 1993-05-10T00:00:00+0000 2.0 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
1GOC 1993-05-10T00:00:00+0000 2.0 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
1JL1 2001-07-13T00:00:00+0000 1.3 D10A E. coli ribonuclease HI
1JL2 2001-07-13T00:00:00+0000 1.76 Crystal structure of TCEO RNase H-a chimera combining the folding core from T. thermophilus RNase H and the remaining region of E. coli RNase H
1JXB 2001-09-06T00:00:00+0000 1.6 I53A, a point mutant of the cysteine-free variant of E. coli Rnase HI

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.9 Ribonuclease HI A6T512 RNH_KLEP7
90.9 Ribonuclease HI B5Y1G2 RNH_KLEP3
92.9 Ribonuclease HI C0Q6N2 RNH_SALPC
93.5 Ribonuclease HI P0A2B9 RNH_SALTY
93.5 Ribonuclease HI P0A2C0 RNH_SALTI
93.5 Ribonuclease HI B4TYH0 RNH_SALSV
93.5 Ribonuclease HI B5BDW5 RNH_SALPK
93.5 Ribonuclease HI A9MZ19 RNH_SALPB
93.5 Ribonuclease HI Q5PFD8 RNH_SALPA
93.5 Ribonuclease HI B4SV39 RNH_SALNS
93.5 Ribonuclease HI B4TK85 RNH_SALHS
93.5 Ribonuclease HI B5R5L3 RNH_SALG2
93.5 Ribonuclease HI B5R449 RNH_SALEP
93.5 Ribonuclease HI B5FJ58 RNH_SALDC
93.5 Ribonuclease HI Q57SZ6 RNH_SALCH
93.5 Ribonuclease HI A9MPF1 RNH_SALAR
93.5 Ribonuclease HI B5F8X2 RNH_SALA4
93.5 Ribonuclease HI A8AKR0 RNH_CITK8
99.4 Ribonuclease HI Q0TLC3 RNH_ECOL5
99.4 Ribonuclease HI A7ZWF6 RNH_ECOHS
99.4 Ribonuclease HI B7LHC0 RNH_ECO55
99.4 Ribonuclease HI B7UJB0 RNH_ECO27
100.0 Ribonuclease HI Q3Z5E9 RNH_SHISS
100.0 Ribonuclease HI P0A7Y7 RNH_SHIFL
100.0 Ribonuclease HI Q32JP9 RNH_SHIDS
100.0 Ribonuclease HI Q325T2 RNH_SHIBS
100.0 Ribonuclease HI B2U352 RNH_SHIB3
100.0 Ribonuclease HI B7LW89 RNH_ESCF3
100.0 Ribonuclease HI B1LHM3 RNH_ECOSM
100.0 Ribonuclease HI B6HZS7 RNH_ECOSE
100.0 Ribonuclease HI B7N876 RNH_ECOLU
100.0 Ribonuclease HI P0A7Y4 RNH_ECOLI
100.0 Ribonuclease HI B1IPU4 RNH_ECOLC
100.0 Ribonuclease HI P0A7Y5 RNH_ECOL6
100.0 Ribonuclease HI B1XD78 RNH_ECODH
100.0 Ribonuclease HI C4ZRV1 RNH_ECOBW
100.0 Ribonuclease HI B7M213 RNH_ECO8A
100.0 Ribonuclease HI B7MQ23 RNH_ECO81
100.0 Ribonuclease HI B7NKW4 RNH_ECO7I
100.0 Ribonuclease HI B5Z0I8 RNH_ECO5E
100.0 Ribonuclease HI P0A7Y6 RNH_ECO57
100.0 Ribonuclease HI B7MBJ0 RNH_ECO45
100.0 Ribonuclease HI A7ZHV1 RNH_ECO24
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI