G₆₇₃ could be a novel mutational hot spot for intragenic suppressors of pheS5 lesion in Escherichia coli

Abstract

The pheS5 Ts mutant of Escherichia coli defined by a G293 → A293 transition, which is responsible for thermosensitive Phenylalanyl-tRNA synthetase has been well studied at both biochemical and molecular level but genetic analyses pertaining to suppressors of pheS5 were hard to come by. Here we have systematically analyzed a spectrum of Temperature-insensitive derivatives isolated from pheS5 Ts mutant and identified two intragenic suppressors affecting the same base pair coordinate G673 (pheS19 defines G673 → T673; Gly225 → Cys225 and pheS28 defines G673 → C673; Gly225 → Arg225). In fact in the third derivative, the intragenic suppressor originally named pheS43 (G673 → C673transversion) is virtually same as pheS28. In the fourth case, the very pheS5 lesion itself has got changed from A293 → T293 (named pheS40). Cloning of pheS+, pheS5, pheS5-pheS19, pheS5-pheS28 alleles into pBR322 and introduction of these clones into pheS5 mutant revealed that excess of double mutant protein is not at all good for the survival of cells at 42°C. These results clearly indicate a pivotal role for Gly225 in the structural/functional integrity of alpha subunit of E. coli PheRS enzyme and it is proposed that G673 might define a hot spot for intragenic suppressors of pheS5.

Glu571 of PheT plays a pivotal role in the thermal stability of Escherichia coli PheRS enzyme

“…The pheT 36 mutant (MG Ts36) showed a 30% reduction in the expression of β‐galactosidase at 42 °C relative to its expression at 30 °C. As was expected in the pheS mutant, the β‐gal expression was reduced by 55% . In the wild type as was expected, the expression was unaffected at 42 °C (Fig.…”

“…). Very unexpectedly, all the mutants expected to harbor the mutation(s) in pheS (MG Ts1‐MG Ts20, MG Ts22‐MG Ts 35, MG Ts38) were found to have the point mutation in G 293 base pair co‐ordinate and with the very same G 293 ‐>A 293 transition as was reported to be present in pheS5 that leads to G 98 ‐>D 98 aminoacid change ( and Ponmani and Munavar ). As was assumed MG Ts16 was indeed a double mutant with G 314 ‐>A 314 in pheS gene leading to G 105 ‐>D 105 amino acid change and G 1856 ‐>A 1856 base pair change in the pheT gene leading to G 619 →E 619 amino acid change.…”

“…pheS5 is a very well‐characterized pheS mutation of E. coli and it is well known that it is primarily defective in translation at 42 °C . Ponmani and Munavar during their work on isolation and characterization of intragenic suppressors of pheS5 have shown that the relative viability of pheS5 is in the order of 10 −7 and IPTG‐induced expression of β‐galactosidase gets reduced by 50% in the pheS5 mutant. Since all the four pheT mutants isolated in this study harbored the very same G 1711 →A 1711, transition named as pheT36 , it is proposed that Glu 571 is very vital for thermal stability of PheT product and in turn PheRS enzyme at high temperature.…”

The cost of evolved constitutive lac gene expression is usually, but not always, maintained during evolution of generalist populations

“…Compensatory mutations have long been used in molecular genetic studies as a tool to identify physical and genetic changes that can suppress the effects of a focal mutation, thereby identifying interacting components (Blank et al., 2014 ; Jarvik & Botstein, 1975 ; Kacar et al., 2017 ; Manson, 2000 ; Ponmani & Munavar, 2014 ; van Leeuwen et al., 2016 ). Increasingly, they are also recognized as being important in broadening the scope of evolutionary trajectories a population can follow (Szamecz et al., 2014 ; Zee et al., 2014 ), allowing adaptations to be selected that might otherwise prove to be evolutionary dead‐ends (Covert et al., 2013 ; Harrison et al., 2015 ), and influencing the ability of populations to simultaneously adapt to multiple environments (Melnyk et al., 2017 ).…”

The cost of evolved constitutive lac gene expression is usually, but not always, maintained during evolution of generalist populations

“…Compensatory mutations have long been used in molecular genetic studies as a tool to identify physical and genetic changes that can suppress the effects of a focal mutation, thereby identifying interacting components (Blank et al 2014;Jarvik and Botstein 1975;Kacar et al 2017;Manson 2000;Ponmani and Munavar 2014;van Leeuwen et al 2016). Increasingly, they are also recognized as being important in broadening the scope of evolutionary trajectories a population can follow (Szamecz et al 2014;Zee et al 2014), allowing adaptations to be selected that might otherwise prove to be evolutionary dead-ends (Covert et al 2013;Harrison et al 2015), and influencing the ability of populations to simultaneously adapt to multiple environments (Melnyk et al 2017).…”