Juan P. G. Ballesta

THE GTPASE CENTER PROTEIN L12 IS REQUIRED FOR CORRECT RIBOSOMAL STALK ASSEMBLY BUT NOT FOR SACCHAROMYCES CEREVISIAE VIABILITY

Protein L12, together with the P0/P1/P2 protein complex, forms the protein moiety of the GTPase domain in the eukaryotic ribosome. In Saccharomyces cerevisiae protein L12 is encoded by a duplicated gene, rpL12A andrpL12B. Inactivation of both copies has been performed and confirmed by Southern and Western analyses. The resulting strains are viable but grow very slowly. Growth rate is recovered upon transformation with an intact copy of the L12 gene. Ribosomes from the disrupted strain lack protein L12 but are able to carry out translationin vitro at about one fourth of the control rate. The L12-deficient ribosomes have also a defective stalk containing standard amounts of the 12-kDa acidic proteins P1β and P2α, but proteins P1α and P2β are drastically reduced. Moreover, the affinity of P0 is reduced in the defective ribosomes. Footprinting of the 26 S rRNA GTPase domain indicates that protein L12 protects in different extent residues G1235, G1242, A1262, A1270, and A1272 from chemical modification. The results in this report indicate that protein L12 is not essential for cell viability but has a relevant role in the structure and stability of the eukaryotic ribosomal stalk.

Activities of nucleoprotein particles derived from rat liver ribosome

80-S ribosomes and 60-S subunits from rat liver were treated at increasing KC1 concentrations giving protein-deficient ribosomal particles whose components were analyzed and their activity tested. Most of the activities assayed stand treatment up to KC1 concentrations of around 0.6 M; peptidyl transferase, measured by the fragment reaction, however was 50% inhibited by 0.5 M KC1 in 60-S subunits but not in 80-S ribosomes. Three proteins, L21, L26 and L31, might be implicated in this loss of activity. 60-S subunits forming part of the 80 S ribosome are more resistant to the salt treatment and the pattern of proteins released by the treatment differs from the one obtained from free 60-S subunits, implying perhaps a change of conformation of this subunit upon association to form 80-S couples. According to their resistance to release by KC1 the proteins of the large sub-unit can be divided into three groups: (1) easily removed, including proteins: L1, L11, L17 and L25 in 80-s subunits and in addition, L5, L8, L9, L13, L20, L22, L26, L29, L31 and L32/33 in 60-S subunits; (2) proteins resistant to release by high salt concentrations in 80-S ribosomes as well as in 60-S subunits, namely proteins L3, L14, L27, L36, L40, L41, X1 and X2; (3) the rest of the proteins which are released in a more or less continuous way throughout the treatment. 5 S RNA is not released by KC1 treatment at the concentrations used. The binding sites for the antibiotics trichodermin and anisomycin are affected in a different way by the salt treatment, indicating that they are structurally different.

Proteins associated with rRNA in the Escherichia coli ribosome

Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.

Characterization of two acidic proteins of Saccharomycescerevisiae ribosome

Abstract In Saccharomyces cerevisiae ribosomes two proteins, L44 and L45, of strong acidic character are detected. These proteins, presumably equivalent to bacterial L7 and L12, have been purified and have given a total cross reaction when tested by double immunodiffusion. Reaction with fluorescamine has shown that the amino terminal group of the polypeptide is blocked in protein L44 and free in protein L45. Tryptic analysis of the two proteins shows that three out of nine peptides are in identical position in both patterns, three more are easily related and the last three are clearly different. The data indicate that proteins L44 and L45 are closely related but not totally identical.