RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

RNA–RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA–RNA interactions can involve complementary Watson–Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA–RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem–loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem–loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem–loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem–loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop–loop ‘kissing’ motif. High affinity binding to the other structural motifs in the decoding region were not observed.     

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.     

The use of hydroxylamine cleavage to produce a fragment of ribosomal protein S4 which retains the capacity to specifically bind 16S ribosomal RNA.

In previous reports we have described the isolation of fragments of 30S ribosomal protein S4 using a number of different enzymatic and chemical cleavage techniques. These experiments were designed to determine the region of the protein responsible for 16S RNA recognition. We report here the isolation of two fragments produced by the hydroxylamine cleavage of the asparaginyl-glycyl peptide bond between positions 124 and 125. The purified fragments were chemically identified and tested for RNA binding capacity. The fragment consisting of residues 1-124 retains RNA binding activity and the fragment 125-203 is totally without RNA binding function. These results and previous results strongly suggest that the domain of protein S4 responsible for 16S RNA specific association is within the region consisting of residues 46-124.     

The characterisation of a fragment of ribosomal protein S4 that is protected against trypsin digestion by 16S ribosomal RNA of Escherichia coli.

After mild trypsin treatment of a complex of ribosomal protein S4 and 16S RNA of Escherichia coli, a large homogeneous fragment of the S4 protein was protected against digestion by its RNA binding site. This fragment was isolated and characterised for molecular weight. It was able to rebind specifically to 16S RNA. Preliminary results indicate that protected protein fragments can also be obtained from other proteins that complex specifically with 23S and 5S RNA.     

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

A cloned Drosophila DNA fragment which codes for a 4 S RNA species.

A collection of random Drosophila melanogaster DNA fragments cloned individually in Escherichia coli was screened for the presence of sequences complementary to the 4 S, 5 S and 5.8 S RNA species produced in the D. melanogaster Kc tissue culture line. Four D. melanogaster DNA fragments were found which possessed sequences complementary to the 4 S RNA species but not complementary to the 5 S or 5.8 S RNA. One such cloned fragment (6.81 kilobase in length) was characterized further. It hybridizes in situ to region 22A-C of the left arm of chromosome 2 and does not contain repetitive sequences detectable by renaturation (cot) analysis. This same region was reported earlier by Steffensen and Wimber (Genetics (1971) 69, 163--178) to hybridize in situ to bulk tRNA extracted from D. melanogaster.     

A ribonucleoprotein fragment of the 30 S ribosome of E. coli containing two contiguous domains of the 16 S RNA.

Ribonucleoprotein fragments of the 30 S ribosome of E. coli have been prepared by limited ribonuclease digestion and mild heating of the ribosome in a constant ionic environment. One such fragment has been described previously. A second electrophoretically homogeneous fragment has now been isolated and its RNA and protein moieties have been characterized. It contains the 5' half of the 16 S RNA, encompassing domains I and II except for the extreme 5' terminus and several small gaps. Seven proteins are present: S4, S5, S6, S8, S12, S15 and S20. The RNA binding sites of five of these proteins are known, and all are RNA sequences that are present in the fragment. Published neutron scattering and immuno-electron microscopic data indicate that six of the proteins are clustered together in a cross sectional slice through the center of the subunit. After deproteinization, the RNA moiety gives two bands in gel electrophoresis, one containing domains I and II and the other, essentially only domain II. The former, although larger, migrates faster in gel electrophoresis, indicating that RNA domains I and II interact with each other in such a way as to become more compact than domain II by itself.     

Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.     

Synthesis of the vasoactive intestinal peptide (VIP) I. The C-terminal cyanogen bromide fragment

The C-terminal cyanogen bromide fragment of VIP, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH₂ (all l), was synthesized to provide evidence for the correctness of the sequence proposed by Mutt and Said (1). The synthesis of this hendecapeptide (VIP_18–28) was carried out by coupling Z-Ala-Val-(Z)Lys to (Z)Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH₂. After removal of the protecting groups and purification, the synthetic material was indistinguishable from the natural fragment on paper chromatograms and electropherograms. Their identity was further confirmed by comparison of the products formed on enzymatic hydrolysis.     

A trypsin-resistant fragment from complexes of ribosomal protein S4 with 16-S RNA of Escherichia coli and from the uncomplexed protein.

A fragment of ribosomal protein S4 was prepared by limited trypsin degestion of a specific complex between protein S4 and 16-S RNA. It was characterised for amino acid sequence and the N-terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg-43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA-binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N-terminal region of the protein is proposed and two probable functions of the excised region are given. Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg-46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein-RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.