Blocking of the initiation of protein biosynthesis by a pentanucleotide complementary to the 3' end of Escherichia coli 16 S rRNA.

Hydrogen bonding between the 3' terminus of 16 S rRNA (... C-A-C-C-U-C-C-U-U-A-OH3) and complementary sequences within the initiator region of mRNA may be a crucial event in the specific initiation of protein biosynthesis (Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1342-1346; Steitz, J. A., and Jakes, K. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4734-4738). Using equilibrium dialysis, we have studied the binding of G-A-dG-dG-U (which is complementary to the 3' end of 16 S rRNA and which has been synthesized enzymatically) to initiation factor-free Escherichia coli ribosomes. We have also investigated the effects of the pentanucleotide on initiation reactions in E. coli ribosomes. G-A-dG-dG-U has a specific binding site on the 30 S ribosome with an association constant of 2 x 10(6) M-1 at 0 degrees C. G-A-dG-dG-U inhibits the R17 mRNA-dependent binding of fMet-tRNA by about 70%, both with 70 S ribosomes and 30 S subunits. In contrast, the A-U-G-dependent initiation reaction and the poly(U)-dependent Phe-tRNA binding was not affected by the pentanucleotide with both ribosomal species.     

Primary sequence of the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.     

16S ribosomal RNA of Escherichia coli contains a N2-methylguanosine at 27 nucleotides from the 3'' end

The 49 nucleotides fragment derived from the 3' end of 16S rRNA by cloacin DF13, is not cleaved by ribonuclease T1 at a guanosine residue tha is present at 27 nucleotides from the 3' terminus (position 115 in 16S rRNA). Analysis of the isolated nucleotide indicates that it is a modified G residue. In vivo labeling with (3H)methionine shows that this G is methylated and co-chromatography with markers reveals that it is N2-methylguanosine.     

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. II. NMR solution structure

The solution structure of the conserved 690 hairpin from Escherichia coli 16 S rRNA was determined by NMR spectroscopy. The 690 loop is located at the surface of the 30 S subunit in the platform region and has been implicated in interactions with P-site bound tRNA, E-site mRNA, S11 binding, IF3 binding, and in RNA-RNA interactions with the 790 loop of 16 S rRNA and domain IV of 23 S rRNA. The structure reveals a novel sheared type G690.U697 base-pair with a single hydrogen bond from the G690 amino to U697-04. G691 and A696 also form a sheared pair and U692 forms a U-turn with an H-bond to the A695 non-bridging phosphate oxygen. The sheared pairs and U-turn result in the continuous single-stranded stacking of five residues from 6693 to U697 with their Watson-Crick functional groups exposed in the minor groove. The overall fold of the 690 hairpin is similar to the anticodon loop of tRNA. The structure provides an explanation for chemical protection patterns in the loop upon interaction with tRNA, the 50 S subunit, and S11. In vivo genetic studies demonstrate the functional importance of the motifs observed in the solution structure of the 690 hairpin.     

Maturation of the 3' end of 5-S ribosomal RNA from Escherichia coli

The 3' ends of 5-S rRNA isolated from Escherichia coli cells were analyzed and identified after different durations of labeling with 32Pi, with and without blocking of protein synthesis. These experiments suggest that the 5-S rRNA starts as a species containing 126 nucleotides, three at each end, and that the extra nucleotides are removed from the 5' and 3' ends in parallel at comparable but different rates. Inhibition of protein synthesis with chloramphenicol blocks, in addition to the 5'-end maturation, the trimming of the extra nucleotides from the 3' end. The trimming of extra nucleotides from both ends of the 5-S rRNA is also affected by the structure of the molecular stalk of 5-S rRNA. A number of observations suggest that the trimmings from both ends are independent processes, which are carried out probably by different enzymes.     

Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit.

The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg²⁺, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle.     

Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits.

The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed.     

Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli

[3H]Dihydrostreptomycin was cross-linked to the 30S ribosomal subunit from Escherichia coli with the bifunctional reagent nitrogen mustard. The cross-linking primarily involved the 16S RNA. To localize the site of cross-linking of streptomycin to the 16S RNA, we hybridized RNA labeled with streptomycin to restriction fragments of the 16S RNA gene. Labeled RNA hybridized to DNA fragments corresponding to bases 892-917 and bases 1394-1415. These two segments of the ribosomal RNA must be juxtaposed in the ribosome, since there is a single binding site for streptomycin. This region has been implicated both in the decoding site and in the binding of initiation factor IF-3, indicating its functional importance.     

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).     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-s RNA in the absence of ribosomal protein. These data yield important information on the topography and organisation of the 5' third of the 16-s RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA.     

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.     

Escherichia coli mRNAs with strong Shine/Dalgarno sequences also contain 5' end sequences complementary to domain # 17 on the 16S ribosomal RNA

A well-established feature of the translation initiation region, which attracts the ribosomes to the prokaryotic mRNAs, is a purine rich area called Shine/Dalgarno sequence (SD). There are examples of various other sequences, which despite having no similarity to an SD sequence are capable of enhancing and/or initiating translation. The mechanisms by which these sequences affect translation remain unclear, but a base pairing between mRNA and 16S ribosomal RNA (rRNA) is proposed to be the likely mechanism. In this study, using a computational approach, we identified a non-SD signal found specifically in the translation initiation regions of Escherichia coli mRNAs, which contain super strong SD sequences. Nine of the 11 E. coli translation initiation regions, which were previously identified for having super strong SD sequences, also contained six or more nucleotides complementary to box-17 on the 16S rRNA (nucleotides 418-554). Mutational analyses of those initiation sequences indicated that when complementarity to box-17 was eliminated, the efficiency of the examined sequences to mediate the translation of chloramphenicol acetyltransferase (CAT) mRNA was reduced. The results suggest that mRNA sequences with complementarity to box-17 of 16S rRNA may function as enhancers for translation in E. coli.