A study of the proteins of nascent and mature mammalian ribosomes

Shepherd, James (1972) A study of the proteins of nascent and mature mammalian ribosomes. PhD thesis, University of Glasgow.

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HeLa cell ribosomal subunits were characterised after dissociation with EDTA or 0.85M KCl. EDTA dissociated subunits, separated on sucrose gradients, showed minimal cross-contamination. Conversely, although the 40s KCl-dissociated subparticle was obtained pure from sucrose gradients, its 60s counterpart was contaminated to 7% with small subunit (AOs) material. CsCl equilibrium centrifugation and isotopic studies of the subparticles derived by EDTA or KCl treatment indicated that although the respective small subunits had identical RNA/protein ratios, KCl dissociated large subunits contained approximately 30% less protein than their EDTA-derived counterpart. It was concluded that part of the protein component of the large ribosomal subunit is loosely associated with that particle and is readily stripped by increasing the ionic strength. II. 45s and 32s RNA was .extracted from HeLa cell nucleolar 80s and 55s particles and was shovm to contain polynucleotide sequences destined to appear on (28s + 18s) and 28s rRNA respectively. The 55s particle, isolated on sucrose gradients, was pure and undegraded. Conversely, 80s material was unstable, and on centrifugation, underwent degradation to particles of sedimentation coefficient 56s and 27s. This degradation could be inhibited completely by treatment of the particles with glutaraldehyde. Recentrifugation of glutaraldehyde fixed 80s particles demonstrated that they were approximately 25% contaminated with 55s material. -Modification of the method of nucleolar particle preparation and of the cells' environment prior to harvesting failed to reduce this degree of contamination and did not improve the yield of 80s material. III. A two dimensional fingerprinting technique was used to characterise the proteins of large and small ribosomal subunits and their putative nucleolar precursors in HeLa cells. It was found that: (a) L-[35S]methionine- or cystine-labelled HeLa cell large and small subunit peptide fingerprints were markedly and reproducibly different from each other; and patterns derived from HeLa monolayer or suspension cell ribosomal subunits presented the same characteristic features. (b) Ribosome dissociation with EDTA or 0.85M KCl did not influence materially the fingerprint pattern for each subunit, nor did variations in the method of subunit protein extraction and processing affect the fingerprints obtained. (c) 55s nucleolar particles contained most peptides found on 50s ribosomes, but no 30s peptides. 80s particles also contained most 50s peptides and, in addition, a few 30s peptides present in much less than molar amounts. On no occasion did the 80s particle peptide fingerprint demonstrate all characteristic 30s spots, although many such peptides Xi7ere identifiable at the top of the sucrose gradient employed to isolate the nucleolar particles. It was concluded that 30s peptide deficiency on 80s fingerprints either reflected the true in vivo situation or resulted from the extraction procedures employed in the isolation of the particles. All "pulse-labelled" peptides on 80s and 55s nucleolar particles could be "chased" onto (50s + 30s) and 50s ribosomal subunits respectively, confirming the precursor-product relationship between these particles. (d) Inhibition of ribosome maturation by the use of actinomycin D or toyocamycin did not prevent the specific association of certain cytoplasmic proteins with prerexisting large ribosomal subunits. The small ribosomal subunit did not participate in a similar particle- protein interaction. (e) The kinetics of appearance in the cytoplasm of ribosome- associated newly synthesised subunit proteins was investigated. After a "pulse" of [35S]methionine, the first labelled proteins to appear on large ribosomal subunits were the "exchangeable" proteins described above. Radioactivity was not found in all 50s subunit proteins until after approximately 60 minutes' labelling, whereas all small subunit proteins were labelled within about 30 minutes. IV. Buoyant density studies of nucleolar and cytoplasmic ribonucleoprotein particles showed that there is progressive protein loss from the nucleolar precursors during their maturation to cytoplasmic ribosomal subunits. 'Moreover, there did not appear to be sequential protein addition to the 80s and 55s particles during their maturation in the nucleolus. On the contrary, an exchange of proteins was apparent between these particles and a nucleolar protein "pool". Furthermore, a 70s particle was found to be a transient intermediate in the 80s to 55s ribonucleoprotein conversion step. V. The two dimensional fingerprinting technique was used in a comparative study of the proteins of ribosomal subunits derived from a number of different eukaryotic species. VI. The results of this study, together with data from the literature, have permitted the formulation of a tentative mechanism of ribosome assembly in HeLa cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: B EH Maden
Keywords: Biochemistry
Date of Award: 1972
Depositing User: Enlighten Team
Unique ID: glathesis:1972-73253
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: https://theses.gla.ac.uk/id/eprint/73253

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