Molecular Genetic Analysis of Mutations That Alleviate rpsL31 Antisuppression in Escherichia coli Work done by REG. NO. A841014 VAIJAYANTHI KANDADAI RAGHAVAN Under the guidance of Prof. Hussain Munavar Department of Molecular Biology School of Biological Sciences Centre for Excellence in Genomic Sciences Madurai Kamaraj University (University with Potential for Excellence) Madurai - 625021 India 19 th APRIL 2010
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Molecular Genetic Analysis of Mutations That Alleviate rpsL31 Antisuppression
in Escherichia coli
Work done by REG. NO. A841014
VAIJAYANTHI KANDADAI RAGHAVAN
Under the guidance of Prof. Hussain Munavar
Department of Molecular BiologySchool of Biological Sciences
Centre for Excellence in Genomic SciencesMadurai Kamaraj University
(University with Potential for Excellence)Madurai - 625021
India
19th APRIL 2010
Previous findings• rpsL encodes the ribosomal protein S12.
Traub, P., and M. Nomura, Science (1968)
• A lesion in rpsL( rpsL31) confers streptomycin resistance, also restricts suppression ( causes antisuppression)mediated by termination suppressors like supE44 (amber suppressor)
Sullivan, M.A., and R.M. Bock, J.Bacteriol. (1985)
• Streptomycin an aminoglycoside, increases misreading.Julian Davies, Walter Gilbert, and Luigi Gorini, PNAS (1964)
Role of supE44 and effect of rpsL31
• supE an Amber suppressor –suppresses amber mutations in Streptomycin sensitive strain
• supE (glnV) encodes a glutamine tRNA• rpsL31 restricts this suppression
Background of the study• Previous studies from the laboratory have classified the
auxotrophic mutations of AB1157 into leaky and tight mutations. • Novel mutation identified, post plating mutagenesis (ppm) , it
increases intrinsic leakiness of markers, was mapped at 74 minutes on the E.coli genome.
(Jayaraman, 1995).
• Recent studies from the lab have shown that ppm which contributes to leakiness and supE44 , the amber suppressor, both are restricted rpsL31 mutation
(Singaravelan, 2009. Ph.D. thesis).
Present workOBJECTIVES
• To isolate mutants that can overcome the restriction posed by rpsL31 on supE44(glnV44) mediated Amber suppression
• Genetic and Molecular characterization of the mutations which counteract rpsL31 restriction.
The Necessity of the study
• To understand pathways and mutations that mediate nonsense suppression and misreading even when misreading/suppression is restricted.
• Can such a pathway affect resistance to streptomycin/other aminoglycosides?
• This understanding of termination suppression has now been extrapolated to curing diseases like Hemophilia
Paula D. James, and David Lillicrap, 2005. Aminoglycoside suppression of nonsense mutations in severe Hemophilia. Blood Journal .106: 3043-3048
Radmila Manev and Hari Manev , Aminoglycoside antibiotics and autism: a speculative hypothesis . BMC Psychiatry
Strain Relevant genotype Source/ Reference
AB1157 hisG4 ∆(gpt-proA) leuB6 thrB1 argE3 rpsL31 glnV44Berlyn MKB,CGSC, USA
• The cold sensitivity conferring mutation could possibly be in the essential genes coding for ribosomal proteins rplQ- rpsM operon, rpsN-rpsH operon
• Some of the proteins coded by these operons are involved in the m-RNA decoding, they mediate interaction of the codon-anticodon pairs ( Potapaov, 1988.) & ( Ramakrishnan, 2000) .
Genes coding for protein present in the Decoding centre
It to be noted that S12 ( rpsL) is closest to the decoding centre
Model proposed Genes coding for
ribosomal proteins of the decoding centre, in their
allelic forms could code for proteins which can allow misreading even
when misreading/non-
sense suppression is restricted through
interactions with the protein S12.
Taken from Ogle et al., 2000.
Future prospects of the study
• Complementation studies to locate srs by cloning
• Characterization of the mutations of the other phenotypic classes of mutants.
• Biochemical analysis to reveal effects of mutation on polysome formation, decoding interactions.
ACKNOWLEDGEMENTS
• Prof. M.Hussain Munavar• Sr.Prof. P. Gunasekaran & Prof. Sripathi Kandula• Mr. B. Singaravelan• CEGS for the instumentation facility and financial
assistance.• CGSC for the E. coli strains.• All the members at Laboratory of Molecular Biology
KEY REFERENCES
1.Anetta N., E. Grzesiuk, 2000. Reversion of argE3 ochre strain Escherichia coli as a tool for studying the stationary phase mutations. Acta Biochemica Polonica. 47:459-4692.Enrico Gallucci, Guido Pacchetti and Sandro Zangrossi, 1970. Genetic studies on temperature sensitive nonsense suppression. Molecular and General Genetics. 106: 362--3703.Gudmundur Eggerrtsson and Dieter soll, 1988. Transfer Ribonucleic acid-Mediated Supression of termination codons in Escherichia coli. Microbiological rev.52:354-3734.Gorini L.,1970. Ribosomal discrimination of tRNAs. Nature New Biol.234:261-2645.Haritha V., J.Philip, 2009. Accuracy modulating mutations of ribosomal protein S4-S5 interface do not necessarily destabilize protein interactions.RNA.15:1100-11096.Kirthi N., Teresa Kelly, 2006. A novel single Amino acid change in Small subunit ribosomal protein S5 has profound effects on translational fidelity. RNA.12:2080-20917. Jayaraman R, 1995. Leakiness of genetic markers and susceptibility to post plating mutagenesis in Escherichia coli. J.Genet.74:85-978.Rosa Nagel, Ana Chan, 2006. Mistranslation and genetic variability the effect of Streptomycin, Mutation research.601:162-1709.Singaravelan B, Ph.D., thesis 2009. Molecular genetic studies on Suppression and related aspects in Escherichia coli. MKU. 10.Strigni P., Gorini L. 1970. Ribosomal mutations affecting efficiency of amber suppression. J. Mol. Biol. 47: 517-530