Ribosomal protein L3: influence on ribosome structure and function

Abstract

Early studies demonstrated roles for ribosomal protein L3 in peptidyltransferase center formation and the ability of cells to propagate viruses. More recent studies have linked these two processes via the effects of mutants and drugs on programmed -1 ribosomal frameshifting. Here, we show that mutant forms of L3 result in ribosomes having increased affinities for both aminoacyl- and peptidyl-tRNAs. These defects potentiate the effects of sparsomycin, which promotes increased aminoalcyl-tRNA binding at the P-site, while antagonizing the effects anisomycin, a drug that promotes decreased peptidyl-tRNA binding at the A-site. The changes in ribosome affinities for tRNAs also correlate with decreased peptidyltransferase activities of mutant ribosomes, and with decreased rates of cell growth and protein synthesis. In vivo dimethylsulfate (DMS) protection studies reveal that small changes in L3 primary sequence also have significant effects on rRNA structure as far away as 100 A, supporting an allosteric model of ribosome function.

Figure 1. The mak8-1 form of L3 affects the structure of the 25S rRNA

Isogenic wild-type and mak8-1 cells were treated with DMS in vivo, reverse transcriptase extension of [³²P]-labeled primers were performed using the extracted rRNAs as templates, the reactions were separated through 6% urea-polyacrylamide denaturing gels, and visualized by autoradiography. A and B. Autoradiograms of the primer extension reactions using Oligo 25-4 (panel A) and Oligo 25-7 (panel B). Sequencing ladders are reverse labeled so as to reflect the sense strand of 25S rRNA in the indicated 5’ → 3’ directions. DMS concentrations are shown in mM. Specific protected or deprotected bases are indicated. C. Results from panels A and B mapped onto the 2-dimensional structure of yeast 25S rRNA. Shown is a selected portion of the domain V and domain VI region of yeast 25S rRNA. Numbering system employed here begins from the 5’ end of the S. cerevisiae 25S rRNA sequence.

Figure 2. Ribosomes containing mutant forms of L3 have higher affinities for aa- and peptidyl-tRNAs

Ribosomes harvested from cells expressing wild-type or mutant forms of L3 were incubated with excess amounts of either [¹⁴C]Phe -aa-tRNA^Phe (A-site, panels A and C), or [¹⁴C]AcPhe-aa-tRNA^Phe (P-site, panels B and D). A and B: Scatchard plot analyses of tRNA binding with wild-type and mutant ribosomes. K_a is equal to the slope of regression trendline and the X-axis intercept corresponds to the fraction of active ribosomes. C and D: Association constants of ribosomes containing the different mutants of ribosomal protein L3 for aa-tRNA at the A-site ([¹⁴C]Phe -aa-tRNA^Phe, panel C), and for acylated-aatRNA at the P-site ([¹⁴C]AcPhe- aa-tRNA^Phe, panel D).

Figure 3. Ribosomes containing mutant forms of L3 have altered drug sensitivity phenotypes

Mid-log phase cultures of isogenic strains harboring the various plasmid-borne RPL3 alleles were spotted in 10-fold dilutions onto SD-trp alone (No Drug), SD-trp containing sparsomycin (35μg/ml), or SD-trp plus anisomycin (25μg/ml). Cells were grown at 30°C for three days.

Figure 4. Global effects of rpl3 mutants on cell growth and protein synthesis

A. Isogenic RPL3::HIS3 gene knockout strains expressing plasmid-borne wild type RPL3 or mutant rpl3 alleles were diluted in selective medium (SD - trp) to O.D.₅₅₀ = 0.01, and growth rates were determined by monitoring optical densities of aliquots taken at 1 h intervals. B. [³⁵S]methionine was added to mid-logarithmically growing isogenic RPL3::HIS3 strains expressing plasmid borne wild type RPL3 or the indicated rpl3 alleles and samples were harvested at 0 min and at 15 min intervals for 75 min. Incorporation of the [³⁵S]-methionine was monitored by cold trichloroacetic acid precipitation as previously described (33). All time points were taken in triplicate.

Figure 5. Mapping of the L3 mutants within the context of the H. marismortui 50S crystal structure at 2.4 Å

A. Global view orienting L3 (blue) with regard to the large subunit rRNA. Specific regions of 25S rRNA are highlighted in color while the remainder of the molecule is rendered in grey. The highlighted regions are as follows: helix 80 (red), helices 89 - 93 (pink), helix 85 (lavender), and 5S rRNA (purple). Ribosomal protein L5 (yeast L11) is rendered in green. B. Close-up view focusing on the relationships between the particular L3 amino acids and rRNAs of interest. L3 is shown in blue, and the positions analogous to yeast W255, P257, and I282 are indicated. The peptidyltransferase center active site is shown in yellow (yeast A2876; E. coli A2451; H. marismortui 2486). The bases of the 23S rRNA that interact with the 3’ end of the aa-tRNA (yeast G2978; E. coli G2553; H. marismoutui U2588) are shown in green, and those that interact with the 3’ end of the peptidyl-tRNA and P-site (Yeast G2669, G2679; E. coli G2252 and G2253; H. marismortui G2284, G2285) are in red. Specific bases whose reactivity to DMS was altered in mak8-1 cells in vivo are outlined in black with the exception of G2978.