Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3' terminal cloacin fragment of 16 S rRNA from nuclease S1

Footprinting of ribosomal protein S1 on the 49-nucleotide 3' terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1.     

Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 3' terminal colicin fragment of Escherichia coli 16S ribosomal RNA. Conformational details revealed by enzymic and chemical probing

The conformation of the colicin fragment of E. coli 16S rRNA was probed with various nucleases and with the adenosine-specific reagent diethylpyrocarbonate (DEP). The results confirm the presence of a stable central hairpin in the colicin fragment and a weaker additional secondary structure involving the regions 5' and 3' to this hairpin. By monitoring DEP accessibility at various stages of heat-denaturation sequential unfolding of individual base pairs was followed. In agreement with previous results it could be shown that dimethylation of the two adjacent adenosines in the hairpin loop (a feature in virtually all ribosomes) leads to a destabilization of the hairpin helix. Accessibilities of G residues, involved in the weaker additional secondary structure is anomalous. One G residue is sensitive to the single strand specific RNase T1 and insensitive to DEP, while the situation is reversed for the adjoining G residue. The strong reaction of the latter G-residue with DEP is unusual and indicates a very special conformation.     

Preparation and sequencing of the cloacin fragment of Streptomyces aureofaciens 16S RNA

A fast method for isolation of a 3'-terminal fragment of Streptomyces aureofaciens 16S RNA was developed. The procedure involves reaction of 70S ribosomes with cloacin DF13 and subsequent fractionation of the reaction mixture by polyacrylamide gel electrophoresis. The cloacin fragment was eluted from the gel and used directly for 3'-end labeling with cytidine-3',5'-[5'-32P]bisphosphate. The labeled RNA fragment was sequenced by the enzymatic method. It consists of 50 nucleotides and has the sequence 5'-GUCGUAACAAGGUAACCGUACCGGA-AGGUGCGGUUGGAUCACCUCCUUUCOH. The differences from the E. coli and Bacillus sequences and their possible influence on the rate and specificity of polypeptide synthesis are discussed.     

The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit

The conformation of the Escherichia coli initiator tRNA has been investigated using enzymatic and chemical probes. This study was conducted on the naked tRNA and on the tRNA involved in the various steps leading to the formation of the 30 S.IF-2.GTP.fMet-tRNA.AUG complex. A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe: (i) the anticodon arm is more rigid; (ii) the presence of an additional nucleotide in the D loop results in specific features in both T and D loops; (iii) C1 and A72 might form a noncanonical base pair. Aminoacylation and formylation induce subtle conformational adjustments near the 3' end, the T arm and the D loop. Initiation factor (IF) 2 interacts with a rather limited portion of the tRNA, covering the T loop and the minor groove of the T stem, and induces an increased flexibility in the anticodon arm. The specific structural features observed in the T loop are probably recognized by IF-2. In the 30 S.IF-2.GTP.fMet-tRNA.AUG complex, additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site.     

Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli

The complete range of published sequences for ribosomal RNA (or rDNA), totalling well over 50,000 bases, has been used to derive refined models for the secondary structures of both 16S and 23S RNA from E. coli. Particular attention has been paid to resolving the differences between the various published secondary structures for these molecules. The structures are described in terms of 133 helical regions (45 for 16S RNA and 88 for 23S RNA). Of these, approximately 20 are still tentative or unconfirmed. A further 20 represent helical regions which definitely exist, but where the detailed base-pairing is still open to discussion. Over 90 of the helical regions are however now precisely established, at least to within one or two base pairs.     

An experimentally-derived model for the secondary structure of the 16S ribosomal RNA from Escherichia coli

Ribonucleoprotein fragments are isolated by mild ribonuclease digestion of E. coli 30S ribosomal subunits, and are deproteinized and subjected to a second partial digestion. Base-pairing between the resulting small RNA fragments is investigated using the two-dimensional gel electrophoresis procedure already reported (see Ref. 1). The interactions thus found are incorporated into a secondary structure model covering approximately 80% of the 16S RNA.     

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.     

Extensions of the known sequences at the 3'' and 5'' ends of 23S ribosomal RNA from Escherichia coli, possible base pairing between these 23S RNA regions and 16S ribosomal RNA.

Extensions of the known sequences at both 3' and 5' ends of 23S ribosomal RNA are presented: The 5' terminal is pG-G-U-U-A-A-G-Cp or pG-G-U... G-U-U-A-A-G-Cp, with a very short sequence between Up and Gp and the 3'terminal is G-A-A-C-C-G-A-(G)-G-C-U-U-A-A-C-C-U-UOH. These two terminal regions exhibit a high degree of complementarity. In addition, extensive complementarities are also found between the 5'terminal sequence of 23S RNA and a sequence contained in section A of the 16S ribosomal RNA, and between the 3'terminal sequence of 23S RNA and sequences in sections O and J in the 16S RNA. The degree of complementarity between the two extremities of 23S RNA, and between these extremities and regions of the 16S RNA, is far greater than would be expected on a random basis suggesting a possible involvement of this base-pairing in the functioning of ribosomes. This possibility is discussed.     

Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure.

Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.     

Determining the polarity of the map of crosslinked interactions in Escherichia coli 16 S ribosomal RNA.

Hydroxymethyltrimethylpsoralen crosslinked 16 S rRNA from Escherichia coli has been R loop hybridized to two plasmid DNAs containing different sections of the 16 S ribosomal gene. It is possible to identify crosslinked features in the part of the RNA that is not complementary to the DNA. Crosslinked features can be aligned into a relative map of interactions. Crosslinked loops that correspond to features located, originally arbitrarily, in the left part of this map are seen in the 5′ half of the 16 S rRNA in one hybrid and loops that correspond to features in the right part of the map are seen in the 3′ two-thirds of the 16 S rRNA in the other hybrid. These results confirm the relative orientations of the crosslinked loops and establish that the left end of the map corresponds to the 5′ end of the molecule.