Analysis of ribosome assembly in a L6 suppressor strains.

<p>The ribosome profiles of <i>rbgA</i>-F6A suppressor strains show an accumulation of a novel 44S complex. Ribosome profiles were analyzed from RB247 (wild-type cells), RB1043 (RbgA-F6A mutant), RB1051 (<i>rbgA</i>-F6A, <i>rplF</i>-R70P), RB1055 (<i>rbgA</i>-F6A, <i>rplF</i>-R3C), RB1057 (<i>rbgA</i>-F6A, <i>rplF</i>-H66L), RB1063 (<i>rbgA</i>-F6A, <i>rplF</i>-G5C), RB1065 (<i>rbgA</i>-F6A, <i>rplF</i>-G5S) and RB1068 (<i>rbgA</i>-F6A, <i>rplF</i>-T68R). Profiles were generated by sucrose density gradient centrifugation. Dashed lines indicate the migration of the 70S, 44S and the 30S subunits in the gradient.</p

Proposed model for the role of RbgA in promoting late-stage large ribosome subunit assembly.

<p>A late assembly intermediate (LAI_50-1) can proceed via two different pathways. Pathway 1 posits that RbgA binds prior to L6 (LAI_50-2) while pathway 2 indicates L6 binds prior to RbgA (LAI_50-3). When bound together (LAI_50-4), RbgA facilitates proper interaction between L6 and the maturing ribosome, which triggers the incorporation of late ribosomal proteins. Once proper incorporation occurs, RbgA leaves the complex. The role of GTP hydrolysis in the assembly process is discussed in the text.</p

<i>In vitro</i> maturation of large subunit intermediates.

<p><b>(A) <i>in vitro</i> maturation of 44S intermediate from RB1055.</b> Ribosome profile from cell lysate of strain RB1055 expressing mutated L6 protein (R3C) and RbgA-F6A protein after incubation at 0°C (blue) and 37°C (red) for 60 minutes. <b>(B) </b><b><i>in vitro</i></b><b> maturation of 45S intermediate from RB1043.</b> Ribosome profiles from cell lysate of strain RB1043 expressing RbgA-F6A protein and wild-type L6 protein after incubation at 0°C (blue) or 37°C (red) for 60 minutes. The X-axis indicates the direction of the profiles from the bottom of the gradient (43%) to the top of the gradient (18%). The Y-axis depicts absorbance at 260 nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the 70S, 50S, 44S and the 30S complexes in the gradient.</p

Composition of in vitro matured particles.

<p>Protein occupancy in 44/45S and 70S samples normalized to that of L20. Samples are colored in pairs with the 44/45S intermediate in a lighter shade than the 70S particle. Samples from strain 1043 (RbgA-F6A) are on the left (blue, green), those from strain 1055 (RbgA-F6A, L6-RC3) are on the right (orange, red). Semitransparent dots signify unique peptide measurements. The median value is denoted with a larger opaque marker. Protein L6 is highlighted in red. Proteins significantly depleted are colored orange.</p

Mutations in L6 protein affect subunit joining/interaction.

<p>Ribosome profiles of strains expressing mutated L6 protein [representative strain RB1123 (<i>rplF</i>-R70P)]. The X-axis indicates the direction of the profiles from the bottom of the gradient (25%) to the top of the gradient (10%). The Y-axis depicts absorbance at 260 nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the 70S, 50S and the 30S complexes in the gradient.</p

Interaction between L6 protein and the 50S ribosomal subunit.

<p><b>A</b>. Crystal structure of 50S subunit from <i>E. coli</i> (PDB ID:2AW4) with the position of L6 indicated in blue. The location of late binding ribosomal proteins that are missing or highly reduced in the 45S particle are also highlighted (L16 (green), L28 (yellow) and L36 (cyan)} or highly reduced {L27 (orange), L33a (purple), L35 (red). <b>B</b>. L6 (blue) binding region including helix 97 (colored magenta) is shown in a magnified view and the residues mutated in suppressor strains are colored in red at the N terminal of L6 protein.</p

Whole cell protein abundance.

<p>A heat map depicting the normalized ribosomal protein abundance in various RbgA perturbation strains. The rows correspond to strains induced RbgA expression (RB301:1 mM), RbgA depletion (RB301:6 µM), RbgA-F6A (RB1043), and RbgA-F6A with suppressor mutations (RB1051:R70P, RB1055:RC3, RB1057:H66L, RB1068:T68R). To facilitate comparison between conditions, each protein's abundance is normalized to that of L20 and then to the protein's level in strain RB301:1 mM. Protein abundance is colored from white (0.3) to red (1.5). Proteins depleted from the 44/45S particles are labeled in orange. Protein L6 is highlighted in red.</p

Strains used in this study.

<p>Strains used in this study.</p

Phenotype of RB1043 (<i>rbgA</i>-F6A) and suppressor strains. (A) RbgA-F6A mutation causes a severe growth defect.

<p>Strains were grown in LB at 37°C. Strain RB1006, depicted by closed circles, is a control strain with wild type <i>rbgA</i> gene under the control of native promoter at <i>amyE</i> locus. RB1043, depicted by closed triangles, expresses <i>rbgA</i>-F6A gene under the control of native promoter at <i>amyE</i> locus. <b>(B–D)</b><b>Growth of strains on LB plates to compare growth phenotypes.</b> (B) RB1006 (Wild-type control strain), (C) RB1043 and RB1044. RB1044 is an independent isolate that contains the <i>rplF</i>-F6A mutation and has an identical phenotype. (D) Two suppressor strains isolated from RB1043, (RB1051 and RB1068), that partially alleviate the growth defect. All strains were cultured on LB plates at 37°C overnight.</p

Protein composition of 44S and 45S particles. (A) Ribosomal protein occupancy in 70S (top) or 44S/45S (bottom) samples.

<p>Protein occupancy is reported as the corrected ¹⁴N/¹⁵N ratio. To account for differences in the amount of sample analyzed, for each peptide, this ratio is normalized to the median calculated ratio for L20, a protein bound stoichiometrically in all samples. Each sample bore the RbgA F6A mutation as well as additional L6 mutations as noted in the legend. Semitransparent dots represent individual peptide measurements. The median value is indicated with a larger opaque marker. Protein L6 is highlighted in red. Proteins significantly depleted are colored orange. <b>(B) Replicate analysis of selected ribosomal protein occupancy.</b> Median protein occupancy values are reported as a heat map for either 70S or 44S/45S samples, which were analyzed in triplicate using independent biological samples. Protein occupancy ranging from 0 to 1.1 is colored white to red. Row labels are color coded by replicate (black to grey). Column labels are colored as described above.</p