Structural Diversity in Bacterial Ribosomes: Mycobacterial 70S Ribosome Structure Reveals Novel Features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria

Investtiigattiion on riibosome structure and dynamiics:: Mycobactteriium riibosome iin focus

This doctoral study deals primarily with the application of cryo-EM and single particle reconstruction technique to investigate three major aspects of translation machinery in prokaryotes. Kinetic studies indicate that in vivo, protein synthesis and ribosome biogenesis involves interaction of additional factors, capable of enhancing efficient assembly and protein synthesis. The first part of the study deals with the characterization of hitherto unknown association of cellular protein factors with the bacterial ribosome and functional importance of such interactions with the ribosome. Biochemical studies and a 3D cryo-EM map of a ribosome sample purified directly from cell extract revealed strong association of two cellular proteins namely Aldehyde alcohol dehydrogenase (AdhE) and Outer membrane protein C (OmpC) with E. coli 70S ribosome. Additional ribosome associated functions of these two proteins reflect multi-tasking activity of proteins within cell. The second part of the study deals with structural characterization of ribosome from bacteria that are of clinical importance to human health like Mycobacterium. The 12Å resolution 3D cryo-EM map of Mycobacterium smegmatis revealed unique additional structural components in the mycobacterial ribosome. Identification of several rRNA helices particularly a rRNA helix lying close to mRNA exit and extensions of ribosomal proteins around the peptide exit tunnel in M. smegmatis 70S ribosome structure reflects diversity of ribosome structure across bacterial kingdom and involvement of species specific structural elements in fine tuning the process of protein biosynthesis. The third part of the study addresses structure of the largest ribosomal protein S1 and its interaction with ribosomes from E. coli and M. smegmatis. Dynamic ribosome binding nature of S1 protein in a species specific manner has been explored. The study also presents a model of full length S1 protein from E. coli and interprets the organization of the domains and interaction of S1 with mRNA as well as ribosomal components

Intrinsic Molecular Properties of the Protein–Protein Bridge Facilitate Ratchet-Like Motion of the Ribosome

The ribosomal intersubunit bridges maintain the overall architecture of the ribosome and thereby play a pivotal role in the dynamics of translation. The only protein–protein bridge, b1b, is formed by the two proteins, S13 and L5 of the small and large ribosomal subunits, respectively. B1b absorbs the largest movement during ratchet-like motion, and its two proteins reorganize in different constellations during this motion of the ribosome. Our results in this study of b1b in the Escherichia coli 70S ribosome suggest that the intrinsic molecular features of the bridging proteins allow the bridge to modulate the ratchet-like motion in a controlled manner. Additionally, another large subunit protein, L31, seems to participate with S13 and L5 in the formation, dynamics, and stabilization of this bridge

Structural analysis of the <i>Msm</i>50S.

<p>(A) Secondary structure diagram of the <i>M. laprae</i> 23S rRNA (left: 5′ end; right: 3′ end). Locations of extra rRNA helices in mycobacterium are highlighted (orange) and marked in the 5′ 23S rRNA structure (domains I, II, III). (B) Stereo view of the solvent side of <i>Msm</i>50S (blue wire mesh) with the coordinates <i>of E. coli</i> 50S subunit (23S rRNA pale cyan, 5S rRNA deep blue, proteins grey) docked inside. The atomic structure is adopted from the crystal structure of <i>E. coli</i> 70S ribosome (Protein Data Bank ID code 2I2V). Major additional density clusters are highlighted in different colors; steeple, deep pink; H15/H16a, orange; H31a, purple; additional density of L25, yellow; additional densities of proteins around the tunnel exit, green. Landmarks are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031742#pone-0031742-g001" target="_blank">Figure 1</a>.</p

Comparison of the 70S Ribosome from <i>M. smegmatis</i> with the <i>E. coli</i> 70S ribosome.

<p>The cryo-EM map of the <i>Msm</i>70S (A, C) is shown together with the cryo-EM map of the <i>Eco</i>70S (B, D; EMD-1395). The ribosomes are shown from the L1 side (A, and B) and the L7/L12 side (C,and D). Missing extended L7/L12 Stalk is marked in <i>Msm</i>70S with solid triangle (◂). Asterisk (*) marks the missing density in the bottom part of the 30S subunit of <i>Msm</i>70S due to shorter h10 and h17. The location of the S1 and S2 proteins where corresponding densities are largely absent in Msm70S is marked with arrow. The computationally separated small subunit (E) and large subunit (F) of the <i>Msm</i>70S are shown with the P site-bound tRNA (green) from the interface sides. The small subunits are shown in yellow, the large subunits blue in all the panels. Landmarks for the 30S subunit: bk, beak; h, head; pt, platform; sh, shoulder; sp, spur; h44, helix 44 of 16S rRNA. Landmarks for the 50S subunit: CP, central protuberance; L1, L1 protein; st, L7/L12 stalk; sb, L7/L12 stalk base; SRL, sarcin ricin loop; H69, helix 69 of 23S rRNA.</p

Identification of the extra rRNA helices and additional segments of r-proteins in the <i>Msm</i>50S.

<p>Close-up views of the (A) H54 region showing that the steeple (deep pink) emerges from H54, (B) H14 and H16 region displaying that the bifurcated density (orange) is related to these helices, and (C) H31 region identifying the density corresponding to the extra helix (purple), H31a, in the 23S rRNA, are shown in stereo. Density clusters attributable to extra domains of <i>M. smegmatis</i> ribosomal large subunit proteins L4/L17/L22/L29 (D), and L25 (E) are also shown in stereo. <i>T. thermophilus</i> L25 (from pdb code 2J01, chain Z, 3–179 amino acids) protein structure is shown here (magenta) to identify the C-terminal domain. Part of the C-terminal extra domain overlaps with the density cluster attributed to L25 in <i>Msm</i>70S.</p

Structural analysis of the <i>Msm</i>30S.

<p>(A) Secondary structure diagram of the <i>M. tuberculocis</i> 16S rRNA. The helices which are different in mycobacterial 16S rRNA as compared to the <i>E. coli</i> 16S rRNA are marked (shorter, red; longer, cyan). (B) Stereo view of the solvent side of <i>Msm</i>30S (yellow wire mesh) with the docked crystal structure of <i>E. coli</i> 30S subunit (16S rRNA in olive, proteins in grey colour) (pdb code: 2I2U). Major extra density clusters (solid yellow) are shown. Proteins with additional segments are coloured and designated with their names. Density cluster marked with asterisk (*) represents the density corresponding to extra components of h9 and proteins S16, S17. Landmarks are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031742#pone-0031742-g001" target="_blank">Figure 1</a>.</p

Neighbourhood of the steeple structure.

<p>Close-up view of the steeple (deep pink) is shown with its neighbouring ribosomal proteins and rRNA structures in stereo. The <i>Msm</i>70S density map is represented in grey wire mesh with the coordinates of the 30S (Wheat colour) and the 50S (light cyan) subunits docked into the map. Coordinate of mRNA (blue stick model) is taken from a crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031742#pone.0031742-Yusupova1" target="_blank">[46]</a> of the <i>T. thermophilus</i> 70S ribosome (pdb code: 2HGR, chain 1) and aligned to the atomic structures used here. A thumbnail view of the Msm70S is shown on top to orient the reader.</p

List of <i>M. smegmatis</i> ribosomal proteins that are bigger than their <i>E. coli</i> counter parts (proteins more than 12 amino acids (aa) longer are mentioned here).

<p>List of <i>M. smegmatis</i> ribosomal proteins that are bigger than their <i>E. coli</i> counter parts (proteins more than 12 amino acids (aa) longer are mentioned here).</p

Tumor Suppressor p53-Mediated Structural Reorganization of the Transcriptional Coactivator p300

Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number of nonhistone proteins, p53 being the most prominent one. Recruitment of p300 to p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggests that p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined domains, where the structured domains are interlinked with unstructured linker regions. A crystal structure of the central domain of p300 encompassing Bromo, RING, PHD, and HAT domains demonstrates a compact module, where the HAT active site stays occluded by the RING domain. However, although p300 has a significant role in mediating the transcriptional activity of p53, only a few structural details on the complex of these two full-length proteins are available. Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex. The three-dimensional cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT domains occurs upon complex formation, which is likely required for exposing HAT active site and subsequent acetyltransferase activity. Our observation correlates well with previous studies showing that the presence of Bromodomain is obligatory for effective acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism whereby p300, following binding with p53, gets activated