The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).     

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

A conformational switch involving the 915 region of Escherichia coli 16 S ribosomal RNA

A novel alternative conformation, which involves an interaction between the 5' terminal and 915 regions (E. coli numbering), is proposed after a screening of compiled sequences of small subunit ribosomal RNAs. This conformation contains a pseudoknot helix between residues 12-16 and 911-915, and its formation requires the partial melting of the 5' terminal helix and the disruption of the 17-19/916-918 pseudoknot helix of the classical 16 S rRNA secondary structure. The alternate pseudoknot helix is proximal to the binding site of streptomycin and various mutations in rRNA which confer resistance to streptomycin have been located in each strand of the proposed helix. It is suggested that the presence of streptomycin favours the shift towards the alternate conformation, thereby stabilizing drug binding. Mutations which destabilize the novel pseudoknot helix would restrict the response to streptomycin.     

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.     

The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli.

The digestion of E. coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration. One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol. wt. 7,600. There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA. 10% of the RNA could be digested from native 30S subunits. Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions. Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA. The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).     

Protein L16 of the Escherichia coli ribosome: possible role in protein biosynthesis

We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.     

Chemical crosslinking of elongation factor G to the 23S RNA in 70S ribosomes from Escherichia coli

Elongation factor G was crosslinked to the 23S RNA of 70S Escherichia coli ribosomes with the bifunctional, cleavable reagent diepoxybutane (DEB). The EF-G-23S RNA complex was isolated and digested with ribonuclease A. After digestion, an RNA fragment, protected by EF-G was cleaved from the complex and isolated. The nucleotide sequence of this RNA fragment was determined by partial ribonuclease digestion. It proved to be 27 nucleotides long and it could be identified with residues 1055 to 1081 of the nucleotide sequence of E. coli 23S RNA. In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a dramatic decrease in the yield of this complex.     

Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.