Cornelis W. A. Pleij

PseudoBase: a database with RNA pseudoknots

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio. Leiden Univ.nl/ approximately Batenburg/PKB.html. This page will direct the user to a retrieval page from where a particular pseudoknot can be chosen, or to a submission page which enables the user to add pseudoknot information to the database or to an informative page that elaborates on the various aspects of the database. For each pseudoknot, 12 items are stored, e.g. the nucleotides of the region that contains the pseudoknot, the stem positions of the pseudoknot, the EMBL accession number of the sequence that contains this pseudoknot and the support that can be given regarding the reliability of the pseudoknot. Access is via a small number of steps, using 16 different categories. The development process was done by applying the evolutionary methodology for software development rather than by applying the methodology of the classical waterfall model or the more modern spiral model.

Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs.

Various plant viral RNAs possess a 3′ terminus with tRNA‐like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl‐tRNA synthetase and ATP. We have studied the structure of end‐labelled 3′‐terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double‐strand‐specific ribonuclease from cobra venom. The data indicate that the 3′ termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three‐dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three‐dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA‐specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA‐like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.

PseudoBase: structural information on RNA pseudoknots

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio.LeidenUniv.nl/ approximately Batenburg/PKB.html. For each pseudoknot, thirteen items are stored, for example the relevant sequence, the stem positions of the pseudoknot, the EMBL accession number of the sequence and the support that can be given regarding the reliability of the pseudo-knot. Since the last publication, information on sizes of the stems and the loops in the pseudoknots has been added. Also added are alternative entries that produce surveys of where the pseudoknots are, sorted according to stem size or loop size.

Pseudoknots: a new motif in the RNA game

In the last few years a novel RNA folding principle called pseudoknotting has emerged. Originally discovered in noncoding regions of plant viral RNAs, pseudoknots now appear to be a widespread structural motif in a number of functionally different RNAs. These structural elements are part of tRNA-like structures and are involved in folding catalytic sites of ribozymes. They increase the efficiency of ribosomal frameshifting or can serve as specific binding sites for regulatory proteins.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting

Abstract RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49°, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

An approximation of loop free energy values of RNA H-pseudoknots.

A set of free energy values is suggested for RNA H-pseudoknot loops. The parameters are adjusted to be consistent with the theory of polymer thermodynamics and known data on pseudoknots. The values can be used for estimates of pseudoknot stabilities and computer predictions of RNA structures.

The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice.

The structure of the tRNA‐like 3′ terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3′ ‐terminal fragment possessing the tRNA‐like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three‐dimensional model for the tRNA‐like structure is given which is compared with those proposed earlier for the tRNA‐like 3′ termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA‐like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl‐transferase reveals a size of ˜100 nucleotides in agreement with the models proposed.

Properties of the tobacco mosaic virus intermediate length RNA-2 and its translation

The existence of subgenomic RNAs is well established in the case of plant viruses such as tobacco mosaic virus (TMV). However, except for the subgenomic coat protein mRNA, it is not known whether the other subgenomic RNAs have a function in the life cycle of the virus. In search of more information about one of the major subgenomic RNAs-intermediate length RNA-2 or I2 RNA-of TMV, in vitro and in vivo translational studies were performed. The I2 RNA, which codes in vitro for the synthesis of a 30K (K = kilodalton) protein, appears to be uncapped as judged by the need of different in vitro translation conditions for the synthesis of this protein, compared to the conditions required for the synthesis of the 126K and 183K proteins coded by the capped genomic RNA. In vivo a protein migrating in the same position as the 30K protein synthesized in vitro can be detected in infected tobacco leaves. Since this protein occurs transiently early upon infection, whether it is virus-coded or virus-induced, it could have an early function during infection.

tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

ABSTRACT For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3′ poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLSTYMV of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3′ TLSBMV (about 200 nucleotides) with determinants for tyrosylation. We discovered TLSBMV-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLSBMV tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLSBMV in trans. Intriguingly, a subdomain of the TLSBMV could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLSBMV during the BMV infection cycle.

Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

The 5′ UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C⋅C and C⋅A mismatches. In this article, evidence is presented indicating that the 5′ proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C⋅C and C⋅A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5′ proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C⋅C and/or C⋅A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA–protein interactions.