Studies on the carboxyl-terminal amino acid sequence of the chaperonin groel from escherichia coli
Previous work from our laboratory had suggested that the carboxyl-terminus of GroEL limited the ability of this protein to reverse the temperature-sensitive defects of dnaAts mutations in E. coli. This carboxyl-terminus consists of a 13 amino acid string of glycine and methionine residues, a motif t...
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| Format: | Dissertation |
| Language: | English |
| Published: |
ProQuest Dissertations & Theses
01-01-1993
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| Online Access: | Get full text |
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| Summary: | Previous work from our laboratory had suggested that the carboxyl-terminus of GroEL limited the ability of this protein to reverse the temperature-sensitive defects of dnaAts mutations in E. coli. This carboxyl-terminus consists of a 13 amino acid string of glycine and methionine residues, a motif that is highly conserved amongst many of the GroEL homologues sequenced so far. A clone was constructed which expressed a form of GroEL lacking this motif. Surprisingly this clone (which also expressed wild-type groES) was unable to suppress dnaAts mutations when overexpressed, but was able to complement groELts mutations (even when present in single copy). A groE deletion mutant strain was constructed and this too was complemented by the truncated form of groEL. The resulting strain, carrying only truncated groEL, was extensively characterized and found to behave identically to an isogenic strain carrying wild type groEL, in that growth rates, temperature dependence, carbon source utilization, bacteriophage sensitivity, ethanol sensitivity, UV sensitivity and β-lactamase excretion were all found to be identical between the two strains. However, competition experiments involving co-culturing of the strains carrying groEL + groELtr demonstrated an advantage to the cells expressing the wild-type gene when grown at 42oC. The advantage was found to be due to the strain encoding the truncated groEL exiting stationary phase and entering log-phase growth more slowly than cells expressing groEL+. It was also found that strains expressing groELtr are more sensitive to the dye crystal violet, and low levels of some other biocide at 420C. This suggests a role for the GroE proteins in membrane biogenesis and.or maintenance as well as an involvement of the glycine-methionine sequence. |
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