Biomedical subjects

C W Pleij

Publications and source records attributed to C W Pleij.

At least 19 recordsLinked to original sources

PubMed2001

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

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 degrees, 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.

Adenine↗

PubMed2001

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.

Base Sequence↗

PubMed2000

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.

Base Sequence↗

PubMed2000

Structure and variability of the 3' end of RNA 3 of Beet soil-borne pomovirus--a virus with uncertain pathogenic effects.

PCR products representing c. 550 3' terminal bases of Beet soil-borne pomovirus (BSBV) RNA 3 were compared for sources of this virus from all major sugarbeet-growing areas in Germany. In none of these areas conspicious symptoms could be attributed to the presence of BSBV. Single strand conformation polymorphism analyses suggested that the BSBV genome may be very variable. This was confirmed by nucleotide sequence analysis. Each PCR product which was analysed showed sequence differences to others. Even the PCR products obtained from plants grown in the same soil sample were different. The highly variable nature of the BSBV genome is in contrast to the much more conserved nature of the Beet necrotic yellow vein virus genome. By means of the STAR programme a secondary structure was predicted for the 3' end of BSBV RNA 3, in which some areas are highly conserved, whereas others are characterized by a clustering of nucleotide exchanges.

3' Untranslated Regions↗

PubMed2000

In vitro transcription by the turnip yellow mosaic virus RNA polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.

Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3' ACCA end. By mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest DeltaG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.

Alfalfa mosaic virus↗

PubMed1999

Mutation analysis of cis-elements in the 3'- and 5'-untranslated regions of satellite tobacco necrosis virus strain C RNA.

The putative, 3'-terminal stem-loop structure in satellite tobacco necrosis virus strain C (STNV-C) RNA constitutes an essential cis-acting structure for the promotion of negative-strand RNA synthesis and a single-stranded tail is also important. The putative, 5'-terminal stem-loop structure in STNV-C RNA is not essential for productive, plus-strand RNA accumulation but is required for optimal accumulation. Residues 2 and 3 are the minimal cis-acting sequences required for RNA synthesis. The RNA of chimeric mutants, which exchanged 3'- and 5'-untranslated regions between STNV-C and helper tobacco necrosis virus strain D RNAs, accumulated in protoplasts, implying similar replication mechanisms for both RNAs.

3' Untranslated Regions↗

PubMed1999

Molecular characterization of a new furovirus mainly infecting rye.

The complete or almost complete nucleotide sequences were determined for the two RNAs of three different sources of a new furovirus which mainly infects rye and for which the name Soil-borne rye mosaic furovirus (SBRMV) is proposed. The genome organization of this virus is virtually identical to that of Soil-borne wheat mosaic furovirus (SBWMV). However, SBRMV and SBWMV differ considerably in the nucleotide sequences of their genomes and in the derived amino acid sequences of their putative gene products. The sequences of the three sources of SBRMV (two from rye and one from wheat) differ between each other in various parts of their genomes by 1% to 10%. Larger differences were found in the readthrough part of the coat protein readthrough protein. There are no indications that the relationship of the C source from wheat to the other two sources from rye is more distant than the relationship between the two sources from rye. The 3'UTRs of both SBRMV RNAs are remarkable in having a predicted upstream pseudoknot domain (UPD) with seven pseudoknots. The same number was also predicted for the UPD of SBWMV RNA 1 whereas the much shorter UDP of SBWMV RNA 2 may form only four pseudoknots.

3' Untranslated Regions↗

PubMed1999

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.

Computer Simulation↗

PubMed1999

Metastable structures and refolding kinetics in hok mRNA of plasmid R1.

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

Bacterial Proteins↗

PubMed1998

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.

Base Pairing↗

PubMed1998

NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA.

Pseudoknot formation folds the 3' ends of many plant viral genomic RNAs into structures that resemble transfer RNA in global folding and in their reactivity to transfer RNA-specific proteins. The solution structure of the pseudoknotted T arm and acceptor arm of the transfer RNA-like structure of turnip yellow mosaic virus (TYMV) was determined by nuclear magnetic resonance (NMR) spectroscopy. The molecule is stabilized by the hairpin formed by the 5' end of the RNA, and by the intricate interactions related to the loops of the pseudoknot. Loop 1 spans the major groove of the helix with only two of its four nucleotides. Loop 2, which crosses the minor groove, interacts closely with its opposing helix, in particular through hydrogen bonds with a highly conserved adenine. The structure resulting from this interaction between the minor groove and single-stranded RNA at helical junctions displays internal mobility, which may be a general feature of RNA pseudoknots that regulates their interaction with proteins or other RNA molecules.

Amino Acyl-tRNA Synthetases↗

PubMed1998

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Algorithms↗

PubMed1998

Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning.

Helix 2 of the central pseudoknot structure in Escherichia coli 16S rRNA is formed by a long-distance interaction between nt 17-19 and 918-916, resulting in three base pairs: U17-A918, C18-G917and A19-U916. Previous work has shown that disruption of the central base pair abolishes ribosomal activity. We have mutated the first and last base pairs and tested the mutants for their translational activity in vivo , using a specialized ribosome system. Mutations that disrupt Watson-Crick base pairing result in strongly impaired translational activity. An exception is the mutation U916-->G, creating an A.G pair, which shows almost no decrease in activity. Mutations that maintain base complementarity have little or no impact on translational efficiency. Some of the introduced base pair substitutions substantially alter the stability of helix 2, but this does not influence ribosome functioning, neither at 42 nor at 28 degrees C. Therefore, our results do not support models in which the pseudoknot is periodically disrupted. Rather, the central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.

Base Composition↗

PubMed1998

The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.

The complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.

Base Sequence↗

PubMed1998

Genome properties of beet virus Q, a new furo-like virus from sugarbeet, determined from unpurified virus.

Based solely on the information that beet virus Q (BVQ) contains tubular particles, the entire nucleotide sequence of its tripartite genome was determined from unpurified virus in ca. 40 ml crude sap from locally infected Chenopodium quinoa. A starting sequence for RNA 1 was generated using primers corresponding to highly conserved helicase domains in the respective RNAs of furo-, pomo-, peclu-, hordei- and tobraviruses, and was extended by a walking random-primed cDNA approach. The similarity of the 3' ends of furoviral RNAs allowed starting sequences for BVQ RNAs 2 and 3 to be obtained once the 3' end of RNA 1 was known. BVQ RNA 1 encodes a protein with a methyltransferase-like, a variable and a helicase-like region, and for a readthrough protein which, in addition, contains an RNA-dependent RNA polymerase region. RNA 2 carries the coat protein gene, a coat protein read-through protein gene and two additional ORFs which may have arisen by deletions from an originally larger readthrough domain. RNA 3 carries a triple gene block resembling that of several other rod-shaped viruses. The 5' UTRs of the three RNAs have the potential to form a series of hairpins with C-A and C-C mismatches resembling those found in tymoviral RNAs. The 3' ends can be folded into tRNA-like structures which are preceded by a long hairpin-like structure and an upstream pseudoknot domain. BVQ belongs to the recently proposed genus Pomovirus; it shows evolutionary relationships to furoviruses in sensu stricto, peclu-, hordei-, tobra-, tymo-, tobamo-, carla- and potexviruses.

Amino Acid Sequence↗

PubMed1998

Minimal template requirements for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus.

From mutational analysis of the 3'-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drastically, and deletion of only the 3'-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3' ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3' ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated deltaG value should be less than -10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

Base Sequence↗

PubMed1997

The 3'-untranslated region of the alpha2C-adrenergic receptor mRNA impedes translation of the receptor message.

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

3T3 Cells↗

PubMed1997

Acetylation of ribosomal protein S5 affected by defects in the central pseudoknot in 16S ribosomal RNA?

We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C18A in the central pseudoknot of their 16S ribosomal RNA. This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro. The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed. The protein gels also showed the appearance of a satellite band next to S5. This band reacted with anti-S5 antibodies and had a slightly increased positive charge. The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine. Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome.

Acetylation↗