Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

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.     

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.     

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5′-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3′ side, which contains the Shine–Dalgarno sequence. Here, we show by in vitro gp43–RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine–Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator’s hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5′-AC-3′ in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.     

Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments.

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.     

Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface

The MS2 coat protein binds specifically to an RNA hairpin formed within the viral genome. By soaking different RNA fragments into crystals of MS2 coat protein capsids it is possible to determine the X-ray structure of the RNA–protein complexes formed. Here we present the structure to 2.85 Å resolution of a complex between a chemically modified RNA hairpin variant and the MS2 coat protein. This RNA variant has a substitution at the –5 base position, which has been shown previously to be pyrimidine-specific and is a uracil in the wild-type RNA. The modified RNA hairpin contains a pyridin-4-one base (4one) at this position that lacks the exocyclic 2-oxygen eliminating the possibility of forming a hydrogen bond to asparagine A87 in the protein. The 4one complex structure shows an unprecedented major conformational change in the loop region of the RNA, whereas there is almost no change in the conformation of the protein.     

RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function

alpha-Sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 S rRNA. We have examined the requirements for the recognition by alpha-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position that corresponds to 4325. There is an absolute requirement for a helical stem; however, it can be reduced from the 7 base pairs in the natural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition; whereas, the bulged nucleotide does not contribute to identification. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG(sarcin)A leads to loss of recognition by the toxin. We propose that the alpha-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacyl-tRNA and of translocation; that translocation is driven by transitions in the structure of the alpha-sarcin domain RNA initiated by the binding of the factors, or the hydrolysis of GTP, or both; and that to toxin inactivates the ribosomes by preventing this transition.     

Solution structure and functional importance of a conserved RNA hairpin of eel LINE UnaL2

The eel long interspersed element (LINE) UnaL2 and its partner short interspersed element (SINE) share a conserved 3′ tail that is critical for their retrotransposition. The predicted secondary structure of the conserved 3′ tail of UnaL2 RNA contains a stem region with a putative internal loop. Deletion of the putative internal loop region abolishes UnaL2 mobilization, indicating that this putative internal loop is required for UnaL2 retrotransposition; the exact role of the putative internal loop in retrotransposition, however, has not been elucidated. To establish a structure-based foundation on which to address the issue of the putative internal loop function in retrotransposition, we used NMR to determine the solution structure of a 36 nt RNA derived from the 3′ conserved tail of UnaL2. The region forms a compact structure containing a single bulged cytidine and a U–U mismatch. The bulge and mismatch region have conformational flexibility and molecular dynamics simulation indicate that the entire stem of the 3′ conserved tail RNA can anisotropically fluctuate at the bulge and mismatch region. Our structural and mutational analyses suggest that stem flexibility contributes to UnaL2 function and that the bulged cytidine and the U–U mismatch are required for efficient retrotransposition.     

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.     

Specificity of Mnt 'master residue' obtained from in vivo and in vitro selections

Mnt is a repressor from phage P22 that belongs to the ribbon–helix–helix family of DNA binding factors. Four amino acids from the N-terminus of the protein, Arg2, His6, Asn8 and Arg10, interact with the base pairs of the DNA to provide the sequence specificity. Raumann et al. (Nature Struct. Biol., 2, 1115–1122) identified position 6 as a ‘master residue’ that controls the specificity of the protein. Models for the interaction have residue 6 of Mnt interacting directly with position 5 of the operator. In vivo selections demonstrated that protein variants at residue 6 bound specifically to operator mutations at that position. Operators in which the wild-type G at position 5 was replaced by T specifically bound to several different protein variants, primarily hydrophobic residues. The obtained protein variants, plus some others, were used in in vitro selections to determine their preferred binding sites. The results showed that the residue at position 6 influenced the preference for binding site bases predominantly at position 5, but that the effects of altering it can extend over longer distances, consistent with its designation as a ‘master residue’. The similarities of binding sites for different residues do not correlate strongly with common measures of amino acid similarities.     

Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.

The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.     

A new NMR solution structure of the SL1 HIV-1Lai loop-loop dimer

Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1_Lai. NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson–Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.     

The hairpin loop but not the bulged C of the iron responsive element is essential for high affinity binding to iron regulatory protein-1

Vertebrates control intracellular iron concentration principally through the interaction of iron regulatory proteins with mRNAs that contain an iron responsive element, a small hairpin with a bulged C. The hairpin loop and bulged C have previously been assumed to be critical for binding and have been proposed to make direct contact with the iron regulatory proteins. However, we show here that a U or G can be substituted for the bulged C provided that specific nucleotides are also present within internal loops. The K(d), IC(50) and chemical modifications of the iron responsive element variants are similar to the wild-type. Results are more consistent with a role in which the C-bulge functions to orient the hairpin for optimal protein binding rather than to directly contact the protein. Characterization of these novel iron responsive element variants may facilitate the identification of additional mRNAs whose expression is controlled by iron regulatory proteins, as well as provide insight into the nature of a critical RNA-protein interaction.     

RNA recognition site of PP7 coat protein

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.     

RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour

We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacteriophage MS2 coat protein using site-specific substitution of 2′-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem–loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein–RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar.     

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones.

We mapped 359 mutations at 25 positions in synthetic variants of the antigenomic ribozyme of the hepatitis delta agent by analyzing the sequences of 188 cDNA clones. These data were used to identify three features of the ribozyme: highly conserved nucleotides, positions with restricted nucleotide substitutions and three-dimensional relationships between nucleotides. The distribution of mutations at the 25 positions was as follows: G-11 (the eleventh nucleotide from the cleavage site) was mutated in 56 clones; G-12 in 36; U-15 in 33; C-13 in 26; G-28 in 23; C-27 in 21; C-29 in 19; U-26 in 17; C-18 in 14; A-14 in 13; C-16 in 13; C-19 in 12; U-17 in 11; A-20 in 10; G-42 in 9; G-40 in 7; G-41 in 7; C-24 in 6; U-32 in 6; U-23 in 5; C-25 in 4; C-21 in 3; G-30 in 3; G-31 in 3; C-22 in 1. All clones containing a mutation at C-25 had an A at this position, suggesting that the extra cyclic amino group present in adenine and cytosine may function during the cleavage event. Mutations at certain positions were common in simple clones (containing only one or two mutations), while mutations at other positions were over-represented in more complex clones. Both compensatory base changes and co-mutational frequencies were used to identify eight pairs of nucleotides which may interact with each other: G-11 and C-18, G-12 and C-27, C-13 and G-28, C-21 and U-23/C-24, C-21 and G-30, U-23 and G-31/U-32, C24 and G-30, C-27 and G-42. These pairs, which involve some of the most conserved positions in the molecule, suggest interactions among nucleotides previously depicted in open-loop structures. The newly proposed points of contact between pairs of nucleotides are compatible with both the axehead and pseudoknot secondary structural models and were combined with previously proposed Watson-Crick base paired helices to produce two three dimensional models. In both of these, C-25 and C-76 are placed near the cleavage site.     

A study of the thermal unfolding of Escherichia coli phenylalanine transfer RNA by chemical modification at elevated temperatures.

Escherichia coli tRNAPhe was modified by 3 M sodium bisulphite pH 6.0 for 24 h in the temperature range 25 degrees C (x 5 degrees C) to 55 degrees C and in the absence of added magnesium ions. The sites and extents of conversion of cytidines to uridine occurring at each temperature were determined by fingerprinting. The new sites of cytidine modification found at higher reaction temperatures were assumed to arise from breakage of secondary and tertiary structure hydrogen bonds involving cytidine residues. From these data, we conclude that hydrogen bonds within the 'complex core' of the tRNA (including the base pairs G-10 . C-25, C-11 . G-24 and C-13 . G-21 within the dihydrouridine stem and the tertiary structure base pair G-15 . C-48 melt at a lower temperature than the tertiary structure hydrogen bonds between G-19 in the dihydrouridine loop and C-56 in the TpsiC loop.     

Change of RNase P RNA function by single base mutation correlates with perturbation of metal ion binding in P4 as determined by NMR spectroscopy

The solution structures of two 27 nt RNA hairpins and their complexes with cobalt(III)-hexammine [Co(NH₃)₆³⁺] were determined by NMR spectroscopy. The RNA hairpins are variants of the P4 region from Escherichia coli RNase P RNA: a U-to-A mutant changing the identity of the bulged nucleotide, and a U-to-C, C-to-U double mutant changing only the bulge position. Structures calculated from NMR constraints show that the RNA hairpins adopt different conformations. In the U-to-C, C-to-U double mutant, the conserved bulged uridine in the P4 wild-type stem is found to be shifted in the 3′-direction by one nucleotide when compared with the wild-type structure. Co(NH₃)₆³⁺ is used as a spectroscopic probe for Mg(H₂O)₆²⁺ binding sites because both complexes have octahedral symmetry and have similar radii. Intermolecular NOE crosspeaks between Co(NH₃)₆³⁺ and RNA protons were used to locate the site of Co(NH₃)₆³⁺ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop in both mutants, but is shifted 3′ by about one base pair in the double mutant. The change of the metal ion binding site is compared with results obtained on corresponding mutant RNase P RNA molecules as reported by Harris and co-workers (RNA, 1, 210–218).     

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA–CopT complexes suggests that, subsequent to loop–loop contact, helix propagation is prevented. Instead, a fully base paired loop–loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop–loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.