NMR Structure of Bacterial Ribosomal Protein L20: Implications for Ribosome Assembly and Translational Control (original) (raw)
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Nucleic Acids Research, 2007
Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.
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.
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.
Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes
Proceedings of the National Academy of Sciences, 2004
15 N-1 H NMR spectroscopy has been used to probe the dynamic properties of uniformly 15 N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex (Ϸ2.3 MDa), [ 1 H-15 N] heteronuclear single-quantum correlation spectra contain Ϸ100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7͞L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T1) and T1 15 N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7͞L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7͞L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7͞L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis. P rotein synthesis in living systems takes place on the ribosome, a complex macromolecular assembly whose structural and functional properties are rapidly emerging from a powerful combination of electron microscopy (EM) and x-ray crystallography (1, 2). In Escherichia coli, the ribosome is composed of 54 different proteins and three RNA molecules (23S, 16S, and 5S rRNA) This 2.3-MDa complex is termed the 70S ribosome and is made up of two components, the 30S and 50S subunits. The translation of genetic information into functional proteins involves a number of auxiliary factors, many of which are GTPases, including IF2, EF-Tu, elongation factor G (EF-G), and RF3 (2). These molecules bind to overlapping sites on the 50S subunit and regulate the transition of the ribosome through various states on the translational pathway. The binding sites are collectively known as the GTPase-associated region, due to the role of this region in stimulating the GTPase activity of the auxiliary factors.
Molecular cell, 2005
Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.
Structures of the Bacterial Ribosome at 3.5 Å Resolution
Science, 2005
We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.