RNA-protein Binding Sites Research Articles

A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151.

Altering the RNA binding specificity of a translational repressor.

The coat proteins of RNA phages MS2 and GA are specific RNA-binding proteins which function to encapsidate viral RNA and to translationally repress synthesis of the viral replicase. The two proteins have highly homologous amino acid sequences, yet they show different RNA binding specificities, recognizing RNA stem-loop structures which differ primarily in the nucleotide sequences of their loops. We sought to convert MS2 coat protein to the RNA binding specificity of GA through the introduction of GA-like amino acid substitutions into the MS2 coat protein RNA-binding site. The effects of the mutations were determined by measuring the affinity of the coat protein variants for RNA in vitro and by measuring translational repression in vivo. We found five substitutions that affect RNA binding. One dramatically reduces binding of MS2 coat protein to both operators. Three others compensate for this defect by nonspecifically strengthening the interaction. Another substitution accounts for the ability to recognize the differences in the RNA loop sequence.

Identification of an RNA binding site for human thymidylate synthase.

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of approximately 36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.

Domain VI of Escherichia coli 23 S ribosomal RNA : Structure, assembly and function

Domain VI at the 3′ end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. α-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited α-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins.

The U1 small nuclear ribonucleoprotein (snRNP) particle, a cofactor in mRNA splicing, contains nine proteins, six of which are also present in other U snRNPs and three of which are specific to the U1 snRNP. Here we have used a reconstituted human U1 snRNP together with snRNP monoclonal antibodies to define the RNA binding sites of one of the U1 snRNP-specific proteins. When Sm monoclonal antibody (specific for the B', B, and D proteins of U snRNPs) was bound to U1 snRNPs prior to micrococcal nuclease digestion, the same approximately equal to 24 nucleotide fragment of U1 RNA (corresponding to nucleotides 120-143 and termed the "Sm domain") was protected as when no antibody was bound prior to digestion. In contrast, when RNP monoclonal antibody, which reacts with the U1 snRNP-specific Mr 70,000 protein, was bound, additional U1 RNA regions were protected against nuclease digestion. This phenomenon, which we term "antibody-mediated nuclease protection," was exploited to map the position of the Mr 70,000 protein to stem-loop I of U1 RNA. However, there were also sites of Mr 70,000 protein interaction with more 3'-ward regions of U1 RNA, particularly the Sm domain. This indicates that in the three-dimensional structure of the U1 snRNP, the RNP and Sm antigens are in contact with each other. The proximity of the Mr 70,000 protein's RNA binding site (stem-loop I) to the functionally important 5' end of U1 RNA suggests that this protein may be involved in the recognition of, or stabilization of base pairing with, pre-mRNA 5' splice sites.

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30 S ribosomal proteins) were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 Å × 140 Å × 90 Å, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5′ and 3′ termini, the m 7G and m 6 2 A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural “switch”. Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2′- O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a “necklace” in one plane around the “throat” of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure. Some, but by no means all, of the RNA helices that have been proposed to be co-axial in the 16 S RNA can be brought into co-axial positions. Crosslinking data from other authors, in particular intra-RNA crosslinks formed using isolated 16 S RNA as the substrate for crosslinking are, in general, not compatible with our model.

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A "bulged" double helix in a RNA-protein contact site.

The binding of ribosomal protein L18 affects specific nucleotides in Escherichia coli 5S RNA as detected by dimethyl sulfate alkylation and RNase A digestion of the 5S-L18 complex. Most of the affected nucleotides are clustered and localize a site of RNA-protein interaction in and around the defined central helix [Fox, G. E. & Woese, C. (1975) Nature (London) 256, 505-507] of 5S RNA. Chemical carbethoxylation of the native 5S RNA with diethyl pyrocarbonate shows that a striking feature of this region is an unstacked adenosine residue at position 66. We propose that this residue exists as a singly bulged nucleotide extending the Fox and Woese central helix by two base pairs in the E. coli sequence (to positions 16-23/60-68) as well as in each of 61 (prokaryotic and eukaryotic) aligned 5S RNA sequences. In each case, the single bulged nucleotide is at the relative position of adenosine-66 in the RNA sequences. The presence of this putative bulged nucleotide appears to have been conserved in 5S RNA sequences throughout evolution, and its identity varies with major phylogenetic divisions. This residue is likely involved in specific 5S RNA-protein recognition or interaction in prokaryotic and eukaryotic ribosomes. The uridine-65 to adenosine-66 internucleotide bond is protected from RNase A digestion in the complex, and carbethoxylation of E. coli adenosine-66 prior to L18 binding affects formation of a stable RNA-protein complex. Thus, we identify a region of E. coli 5S RNA protected by the ribosomal protein L18 and propose that it contains a bulged nucleotide residue important in stable formation of this RNA-protein complex. This bulged residue appears to be evolutionarily conserved and phylogenetically defined in 5S RNA sequences in general, and consideration of other known RNA-protein binding sites shows that such a "bulged helix" may be a common feature of RNA-protein contact sites.

Small-angle x-ray scattering study of S4-RNA, the 16 S RNA binding site for the protein S4 from Escherichia coli ribosomes

The ribonucleic acid (16 S RNA) of the 30 S Escherichia coli ribosomal subunit binds many of the proteins in this subunit, such as, for instance, S4, S7, S8, S15 and S20 [1,2]. The 16 S RNA binding site for the S4 protein, S4-RNA, can be isolated in pure form [3-91 ; and it is indicated that this binding site consists mainly of two regions of approximately 150 and 160 nucleotides, originating from the 5’ terminal third of the 16 S RNA molecule [7]. These two regions, which are separated by about 120 nucleotides of the 16 S RNA sequence, seem to be stabilized by a specific RNA-RNA interaction [7] . Although the sequence of the S4-RNA region, prepared with both T1 and pancreatic ribonucleases, has been partially analysed [7,9], the correct molecular weight and the size and shape of S4-RNA are not known. We have analysed S4-RNA by using the small-angle X-ray scattering method; the results yield a molecular weight of 136 000 and a radius of gyration of 43.5 A. The X-ray scattering curve can be explained, in its proximal angular range, by the scattering from a twoparameter, uniform electron density model with a shape of an oblate ellipsoid and the dimensions of 132 X 132 X 32 a. 2. Materials and methods

Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli.

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.

Molecular interactions of ribosomal components. 3. Isolation of the RNA binding site for a ribosomal protein.

Molecular interactions of ribosomal components. 3. Isolation of the RNA binding site for a ribosomal protein.