Interaction of R17 Coat Protein With Its RNA Binding Site For Translational Repression

Abstract

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kj at 2°C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of K_a and a ΔH = ?19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RN As failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific.

We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.     

Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site

The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.     

Enzymatic synthesis of a 21-nucleotide coat protein binding fragment of R17 ribonucleic acid

An oligoribonucleotide with a sequence identical with the bacteriophage R17 replicase initiator region has been synthesized. The sequence also encompasses the binding domain of R17 coat protein, which is known to act as a translational repressor at this site. The 21-nucleotide fragment was synthesized entirely by enzymatic methods, T4 RNA ligase being used to join shorter oligomers. The resulting fragment has a secondary structure with the expected thermal stability. Since the synthetic fragment binds R17 coat protein with the same affinity as a 59-nucleotide fragment isolated from R17 RNA, we conclude that it has full biological activity.     

Specific RNA binding by Q beta coat protein

The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0. Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy. The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions. Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop. Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential. A bulged adenosine present in the coat protein binding site was not required for coat protein binding. Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator.     

Spatial determinants of the alfalfa mosaic virus coat protein binding site

The biological functions of RNA-protein complexes are, for the most part, poorly defined. Here, we describe experiments that are aimed at understanding the functional significance of alfalfa mosaic virus RNA-coat protein binding, an interaction that parallels the initiation of viral RNA replication. Peptides representing the RNA-binding domain of the viral coat protein are biologically active in initiating replication and bind to a 39-nt 3'-terminal RNA with a stoichiometry of two peptides: 1 RNA. To begin to understand how RNA-peptide interactions induce RNA conformational changes and initiate replication, the AMV RNA fragment was experimentally manipulated by increasing the interhelical spacing, by interrupting the apparent nucleotide symmetry, and by extending the binding site. In general, both asymmetric and symmetric insertions between two proposed hairpins diminished binding, whereas 5' and 3' extensions had minimal effects. Exchanging the positions of the binding site hairpins resulted in only a moderate decrease in peptide binding affinity without changing the hydroxyl radical footprint protection pattern. To assess biological relevance in viral RNA replication, the nucleotide changes were transferred into infectious genomic RNA clones. RNA mutations that disrupted coat protein binding also prevented viral RNA replication without diminishing coat protein mRNA (RNA 4) translation. These results, coupled with the highly conserved nature of the AUGC865-868 sequence, suggest that the distance separating the two proposed hairpins is a critical binding determinant. The data may indicate that the 5' and 3' hairpins interact with one of the bound peptides to nucleate the observed RNA conformational changes.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.     

RNA binding properties of the coat protein from bacteriophage GA.

The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

RNA binding site of R17 coat protein

The specific interaction between R17 coat protein and its target of translational repression at the initiation site of the R17 replicase gene was studied by synthesizing variants of the RNA binding site and measuring their affinity to the coat protein by using a nitrocellulose filter binding assay. Substitution of two of the seven single-stranded residues by other nucleotides greatly reduced the Ka, indicating that they are essential for the RNA-protein interaction. In contrast, three other single-stranded residues can be substituted without altering the Ka. When several of the base-paired residues in the binding site are altered in such a way that pairing is maintained, little change in Ka is observed. However, when the base pairs are disrupted, coat protein does not bind. These data suggest that while the hairpin loop structure is essential for protein binding, the base-paired residues do not contact the protein directly. On the basis of these and previous data, a model for the structural requirements of the R17 coat protein binding site is proposed. The model was successfully tested by demonstrating that oligomers with sequences quite different from the replicase initiator were able to bind coat protein.     

Specific interaction between the classical swine fever virus NS5B protein and the viral genome.

The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3' untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3' UTR and that the NS5B protein was also able to bind the minus-strand 3' UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3' UTR more efficiently than the plus-strand 3' UTR. Further, we observed that the plus-strand 3' UTR with deletion of CCCGG or 21 continuous nucleotides at its 3' terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3' UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3' terminal. Therefore, it is indicated that the 3' CCCGG sequence of the plus-strand 3' UTR, and the 3' CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3' 21 nucleotide terminal site of both the 3' UTRs.     

Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene.

The interaction in vitro of the RNA of the Q-strain of cucumber mosaic virus (CMV) with its satellite RNA (sat-RNA) has been studied. In hybridisation reactions containing 30% formamide at 45 degrees, sat-RNA binds to CMV RNA 3 and 4 but not to CMV RNA 1 and 2 or RNA from tobacco mosaic virus and alfalfa mosaic virus. The viral coat protein gene present in RNA 3 and 4 contains the site of binding but this region does not contain complementary sequences of any significant length to the sat-RNA sequence. However, the optimum alignment of short complementary sequences present in these regions revealed a stable structure in which it is proposed that sat-RNA twists around the coat protein gene so that two separate blocks of nucleotides in sat-RNA base pair in opposite directions with two adjacent blocks in the coat protein gene to form a knot-like structure. The binding site is a region of 33 nucleotides within the coding region of the coat protein gene which base pairs with residues 98-113 and 134-152 of sat-RNA. The possibility of the binding region of sat-RNA functioning as an "anti-sense" sequence in regulation of the viral coat protein synthesis is discussed.     

Observations concerning the sequence of two additional specifically encapsidated RNA fragments originating from the tobacco-mosaic-virus coat-protein cistron.

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the prededing paper, two other species, fragment 41 and fragment 21 are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, consitute a continuous stretch of 232 nucleotides of the cistron which codes for animo acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21-41-1. A possible model for the secondary structure of this portion of the sequence is proposed.     

A comparison of two phage coat protein-RNA interactions.

The interaction between the coat protein of the group I bacteriophage fr with its translational operator site is compared with the previously studied R17 interaction. The sequence of the two RNA binding sites differ by 2 of 20 nucleotides and two coat proteins by 17 of 129 amino acids. An analysis of the binding of fr coat protein to 24 operator variants revealed that the two proteins recognize operator sequences in virtually the same way. However, fr coat protein binds to nearly every RNA 6 to 14-fold tighter than R17 coat protein. Since the fr operator is a weaker binding variant and the fr coat protein shows a different temperature dependence of binding, it is unlikely that the two systems have different Kas in vivo. RNA fragments containing the operator sequences can initiate the capsid assembly with both fr and R17 coat protein. Surprisingly, the two coat proteins can form a mixed capsid in vitro.     

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.     

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.     

RBPBind: Quantitative Prediction of Protein-RNA Interactions

There are hundreds of RNA binding proteins in the human genome alone and their interactions with messenger and other RNAs in a cell regulate every step in an RNA's life cycle. To understand this interplay of proteins and RNA it is important to be able to know which protein binds which RNA how strongly and where. Here, we introduce RBPBind, a web-based tool for the quantitative prediction of the interaction of single-stranded RNA binding proteins with target RNAs that fully takes into account the effect of RNA secondary structure on binding affinity. Given a user-specified RNA and a protein selected from a set of several RNA-binding proteins, RBPBind computes their binding curve and effective binding constant. The server also computes the probability that, at a given protein concentration, a protein molecule will bind to any particular nucleotide along the RNA. The sequence specificity of the protein-RNA interaction is parameterized from public RNAcompete experiments and integrated into the recursions of the Vienna RNA package to simultaneously take into account protein binding and RNA secondary structure. We validate our approach by comparison to experimentally determined binding affinities of the HuR protein for several RNAs of different sequence contexts from the literature, showing that integration of raw sequence affinities into RNA secondary structure prediction significantly improves the agreement between computationally predicted and experimentally measured binding affinities. Our resource thus provides a quick and easy way to obtain reliable predicted binding affinities and locations for single-stranded RNA binding proteins based on RNA sequence alone.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.