A structural analysis of Xenopus laevis oocyte 5S rRNA

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dc.contributor.authorLeal Carretero, María Luisa Isabelen_US
dc.date.accessioned2024-08-14T21:03:04Z
dc.date.available2024-08-14T21:03:04Z
dc.date.copyright1989en_US
dc.date.issued1989
dc.degree.departmentDepartment of Biochemistry and Microbiologyen_US
dc.degree.levelMaster of Science M.Sc.en
dc.description.abstractThe purpose of this project was to investigate the secondary and tertiary structures of Xenopus laevis oocyte 5S rRNA using chemical and enzymatic probes. Mutations were introduced into the X. laevis 5S rRNA gene in order to study the effect that these mutations would have on the structure of 5S rRNA transcribed from the gene. Four different mutants were constructed by introducing base substitutions in loops D and E of the 5S rRNA structure. Three of the mutants have sequence substitutions in loop E (Xlo73-76, Xlo99-101 and Xlo96-101), and mutant Xlo87-90 has sequence substitutions in loop D. The accessibility of all 5S rRNAs to single-strand specific nucleases (T1,T2,A, and S1), and to a nuclease specific for double-stranded or structured regions (RNase V1), was determined. The reactivity of nucleobases at N7, N3 and Nl positions to chemical probes (DMS, DEPC, and CMCT), was investigated under native (SmM MgCl2, l00mM KCL, 20°C), and semi-denaturing (lmM EDTA, 20°C) conditions. N-ethylnitrosourea was used to identify phosphates not reactive to alkylation under native conditions. The enzymatic accessibility and chemical reactivity results obtained from all four mutants, as well as from the wild-type 5S rRNA, confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. A comparison of the data obtained from the four mutants with that obtained from wild-type 5S rRNA yielded the following results: (i) The conformation of loops A, B, and C is virtually identical in wild-type 5S rRNA and in all four mutants. (ii) In mutants Xlo73-76 and Xlo99-101, region Eis more reactive to chemical probes and more susceptible to enzymatic clevage, and helices IV and V are less stable than the corresponding regions in wild-type 5S rRNA. In Xlo96-101, region E is less reactive, and helices IV and V more stable than their corresponding wild-type regions. (iii) In mutant Xlo87-90, loop D is more susceptible to enzymatic cleavage and more reactive to chemical probes, and helix IV is less stable than the same regions in wild-type 5S rRNA. These results support the conformation of loops€ and Das they appear in the computer graphic model proposed by Westhof et al. (in press) for the tertiary structure of X. laevis oocyte 5S rRNA. In particular, two important features of this model are supported by the structure probing of the four mutants studied here: (i) region E and loop D seem to contain unusual base pairing, consisting of non­-canonical base-pairs; and (ii) no tertiary interactions appear to take place between loop C and either region E or loop D.
dc.format.extent129 pages
dc.identifier.urihttps://hdl.handle.net/1828/18612
dc.rightsAvailable to the World Wide Weben_US
dc.titleA structural analysis of Xenopus laevis oocyte 5S rRNAen_US
dc.typeThesisen_US

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