Analysis of Conformational Changes in 16S rRNA During the Course of 30S Subunit Assembly

2005 ◽  
Vol 354 (2) ◽  
pp. 340-357 ◽  
Author(s):  
Kristi L. Holmes ◽  
Gloria M. Culver
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Subunit Assembly ◽  
30S Subunit

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli

2018 ◽  
Vol 430 (12) ◽  
pp. 1745-1759 ◽  
Author(s):  
Brian A. Smith ◽  
Neha Gupta ◽  
Kevin Denny ◽  
Gloria M. Culver
Keyword(s):  
16S Rrna ◽  
Rrna Processing ◽  
Subunit Assembly ◽  
E Coli ◽  
30S Subunit

scholarly journals Site-Directed Mutants of 16S rRNA Reveal Important RNA Domains for KsgA Function and 30S Subunit Assembly

Biochemistry ◽  
2011 ◽  
Vol 50 (5) ◽  
pp. 854-863 ◽  
Author(s):  
Pooja M. Desai ◽  
Gloria M. Culver ◽  
Jason P. Rife
Keyword(s):  
16S Rrna ◽  
Subunit Assembly ◽  
30S Subunit ◽  
Rna Domains

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

scholarly journals Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate

2019 ◽  
Vol 47 (19) ◽  
pp. 10414-10425 ◽  
Author(s):  
Amal Seffouh ◽  
Nikhil Jain ◽  
Dushyant Jahagirdar ◽  
Kaustuv Basu ◽  
Aida Razi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Catalytic Residue ◽  
Gtp Hydrolysis ◽  
Subunit Assembly ◽  
Cryo Electron Microscopy ◽  
Assembly Factor ◽  
Assembly Intermediate ◽  
Immature Particle

Abstract Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

scholarly journals Irreversible chemical steps control intersubunit dynamics during translation

2008 ◽  
Vol 105 (40) ◽  
pp. 15364-15369 ◽  
Author(s):  
R. Andrew Marshall ◽  
Magdalena Dorywalska ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Large Scale ◽  
Bond Formation ◽  
Elongation Factor ◽  
Resonance Energy ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Gtp Hydrolysis ◽  
30S Subunit

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

Structure and function of ribosomal RNA

1995 ◽  
Vol 73 (11-12) ◽  
pp. 997-1009 ◽  
Author(s):  
Harry F. Noller ◽  
Rachel Green ◽  
Gabriele Heilek ◽  
Vernita Hoffarth ◽  
Alexander Hüttenhofer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Hydroxyl Radical ◽  
Ribosomal Proteins ◽  
23S Rrna ◽  
Active Population ◽  
30S Subunit ◽  
Radical Cleavage ◽  
The Individual ◽  
And Function

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.

scholarly journals 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

2015 ◽  
Vol 59 (5) ◽  
pp. 2807-2816 ◽  
Author(s):  
Miloje Savic ◽  
S. Sunita ◽  
Natalia Zelinskaya ◽  
Pooja M. Desai ◽  
Rachel Macmaster ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Resistance Mechanism ◽  
Binding Pocket ◽  
Product Inhibition ◽  
Sorangium Cellulosum ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
30S Subunit ◽  
Self Protection

ABSTRACTMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of theSorangium cellulosumSo ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum inEscherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of theS-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition byS-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate.

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