The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison, Despite large... Show moreThe secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison, Despite large sequence differences, there are remarkable similarities in the secondary structure, In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches, Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied, UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions, The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour, It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR, Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding. Show less
To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which... Show moreTo examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which changes the central base pair of helix 2, C-18-G(917) to an A(18)xG(917) mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested, Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits, Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control, However, the A(18) mutation has a profound effect on the overall stability of the subunit, The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins, Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins, Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution, Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins, We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex, We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes. Show less
The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of... Show moreThe RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed. (C) 1996 Academic Press, Inc. Show less
Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II... Show moreReplication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm, The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption, The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed. Show less
A procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as... Show moreA procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as disruption of temporarily formed structures, the folding of a molecule during its synthesis and pseudoknot formation. The simulations are able to predict functional metastable foldings and kinetically driven transitions to more stable structures. The analysis of free energies for intermediate foldings allows estimation of the ranges of kinetic refolding barriers and suggests that in some RNAs the selective evolutionary pressure suppresses the possibilities for alternative structures that could form in the course of transcription. It is shown that the folding pathway simulation can result in structure predictions that are more consistent with phylogenetically proven structures than minimum energy solutions. This suggest that RNA folding kinetics is very important for the formation of functional RNA structures. Therefore, apart form its value for predictions of RNA structures, the proposed computer simulations ran be a powerful tool in the studies of RNA folding features. Show less
The possibilities of using a genetic algorithm for the prediction of RNA secondary structure were investigated. The algorithm, using the procedure of stepwise selection of the most fit structures ... Show moreThe possibilities of using a genetic algorithm for the prediction of RNA secondary structure were investigated. The algorithm, using the procedure of stepwise selection of the most fit structures (similarly to natural evolution), allows different models of fitness or driving forces determining RNA structure to be easily introduced. This can be used for simulation of the RNA folding process and for the investigation of possible folding pathways. Such an algorithm needs several modifications before it can predict RNA secondary structures. After modification, a fair number of correct stems are predicted, even when using computationally quick, but very crude, fitness criteria such as stem length and stacking energy, including elements of tertiary structure (pseudoknots). The fact that genetic algorithm simulation includes both stem formations and stem disruption allows one to observe intermediate structures that may be used in combination with phylogenetic or experimental research. Show less
Gultyaev, A.P.; Batenburg, E. van; Pleij, C.W.A. 1994
The secondary structure of satellite tobacco mosaic virus (STMV) RNA was predicted using computer simulations of RNA folding. The analogies of structural elements in the 3' end untranslated regions... Show moreThe secondary structure of satellite tobacco mosaic virus (STMV) RNA was predicted using computer simulations of RNA folding. The analogies of structural elements in the 3' end untranslated regions (3'-UTR) of tobamoviral RNAs were analysed. In addition to the tRNA-like structure and pseudoknot stalk, which are found in all known RNAs of tobamoviruses and STMV, another region of stable consecutive pseudoknots was predicted in the 3'-UTR of STMV RNA. A similar pattern of repeated structural units, containing pseudoknot stalks and parts of the tRNA-like structure, was also found in odontoglossum ringspot virus (ORSV) RNA 3'-UTR. The predictions on the structure are supported by sequence comparisons which point to an important functional role of 3' terminal pseudoknots in STMV RNA as well as in other tobamoviral RNAs. The possible participation of pseudoknotted structures in the interactions with coat protein in STMV is discussed. Show less
An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3'... Show moreAn in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m(2)(6)A1518 and m(2)(6)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition. Show less
Poot, R.A.; Brink, M.F.; Pleij, C.W.A.; Boer, H.A. de; Duin, J. van 1993
We describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which... Show moreWe describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which have an anti-Shine-Dalgarno sequence that is altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded specialized 30S subunits are separated from their chromosomally encoded wild-type counterparts by affinity chromatography that exploits the different Shine-Dalgarno complementarity. An oligonucleotide complementary to the 3' end of wild-type 16S rRNA and attached to a solid phase matrix retains the wild-type 30S subunits. The flow-through of the column contains close to 100% mutant 30S subunits. Toeprinting assays demonstrate that affinity column treatment does not cause significant loss of activity of the specialized particles in initiation complex formation, whereas elongation capacity as determined by poly(Phe) synthesis is only slightly decreased. The method described offers an advantage over total reconstitution from in vitro transcribed mutant 16S rRNA since our 30S subunits contain the naturally occurring base modifications in their 16S rRNA. Show less
The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a... Show moreThe genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E.coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E.coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E.coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leaf roll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent. Show less