Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain (original) (raw)
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Formation of Tertiary Interactions during rRNA GTPase Center Folding
Journal of molecular biology, 2015
The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg(2+) ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg(2+)-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg(2+) addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring fro...
Crystal Structure of a Conserved Ribosomal Protein-RNA Complex
Science, 1999
The structure of a highly conserved complex between a 58-nucleotide domain of large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11 has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA structure that is stabilized by extensive tertiary contacts and contains an unusually large core of stacked bases. A bulge loop base from one hairpin of the RNA is intercalated into the distorted major groove of another helix; the protein locks this tertiary interaction into place by binding to the intercalated base from the minor groove side. This direct interaction with a key ribosomal RNA tertiary interaction suggests that part of the role of L11 is to stabilize an unusual RNA fold within the ribosome.
RNA, 2018
Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide E. coli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent catio...
Nucleic Acids Research, 2005
Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G. A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037-1043 and 1112-1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson-Crick/Watson-Crick (cis W.C.) G. A conformation for the tandem (G. A) 2 in the analog and a minimally perturbed helical duplex stem. Mg 2+ titration studies imply that the cis W.C. geometry of the tandem (G. A) 2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg 2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg 2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg 2+ coordination.
Journal of Molecular Biology, 1997
The three-dimensional solution structure has been determined by NMR spectroscopy of the 75 residue C-terminal domain of ribosomal protein L11 (L11-C76) in its RNA-bound state. L11-C76 recognizes and binds tightly to a highly conserved 58 nucleotide domain of 23 S ribosomal RNA, whose secondary structure consists of three helical stems and a central junction loop. The NMR data reveal that the conserved structural core of the protein, which consists of a bundle of three a-helices and a two-stranded parallel b-sheet four residues in length, is nearly the same as the solution structure determined for the non-liganded form of the protein. There are however, substantial chemical shift perturbations which accompany RNA binding, the largest of which map onto an extended loop which bridges the C-terminal end of a-helix 1 and the ®rst strand of parallel b-sheet. Substantial shift perturbations are also observed in the N-terminal end of a-helix 1, the intervening loop that bridges helices 2 and 3, and a-helix 3. The four contact regions identi®ed by the shift perturbation data also displayed protein-RNA NOEs, as identi®ed by isotope-®ltered three-dimensional NOE spectroscopy. The shift perturbation and NOE data not only implicate helix 3 as playing an important role in RNA binding, but also indicate that regions¯anking helix 3 are involved as well. Loop 1 is of particular interest as it was found to be¯exible and disordered for L11-C76 free in solution, but not in the RNA-bound form of the protein, where it appears rigid and adopts a speci®c conformation as a result of its direct contact to RNA.
Journal of Molecular Biology, 2002
L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50 S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23 S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1 -58 and a C-terminal a-helical domain. This is in contrast with what is observed in the bacterial 50 S subunit, where the N-terminal region folds as an elongated a-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The prefolded C-terminal domain would make a primary interaction with a specific site on the 23 S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.
Journal of Molecular Biology, 1990
Ribonuclease and chemical probes were used Vo investigate the binding sites of ribosomal protein L11 and the pentameric complex L10.(L12)4 on Escherichia coli 23 S RNA. Protein complexes were formed with an RNA fragment constituting most of domains I and II or with 23 S RNA and they were investigated by an end-labelling method and a reverse transcriptase procedure, respectively. The results demonstrate that the two protein moieties bind at adjacent sites within a small RNA region. The Lll binding region overlaps with those of the modified peptide antibiotics thiostrepton and micrococcin and is constrained structurally by a three-helix junction while the L10.(L12)4 site is centred on an adjacent internal loop. The secondary structure of the whole region was determined in detail by the phylogenetic sequence comparison method, and the results for the Lll binding region, together with the experimental data, were used in a computer graphics approach to build a partial RNA tertiary structural model. The model provides insight into the topography of the L11 binding site. It also provides a structural rationale for the mutually cooperative binding of protein Lll with the antibiotics thiostrepton and micrococcin, and with the L10.(L12)4 protein complex. Ribonuelease Specificity CV N~p double-stranded T, Gp~ single-stranded T2 Ap~ >Np~ single-stranded Chemical Reactive atoms DMS G(N-7) > A(N-1) > C(N-3) DEP A(N-7) >>G(N-7)
Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly
Nature structural & molecular biology, 2015
We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.