Institut Jacques Monod CNRS, Universités Paris 6-7, Laboratoire Génétique et Membranes, Tour 43, 2 place Jussieu 75251, Paris Cedex 05, France1
Author for correspondence: Marie-Françoise Petit-Glatron. Tel: +33 1 44 27 47 19. Fax: +33 1 44 27 59 94. e-mail: glatron{at}ccr.jussieu.fr
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ABSTRACT |
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Keywords: mRNA decay, exocellular proteins, sacR, 5' mRNA stabilizer
Abbreviations: SD, ShineDalgarno
a Present address: Department of Plant Pathology, University of California, Davis, California 95616, USA.
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INTRODUCTION |
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To test this hypothesis we assessed the transcriptional efficiency of the gene fusions and the stability of the resulting transcripts. This comparative study was enlarged to include chitosanase, a native extracellular protein that is produced at a low level during the exponential growth phase in B. subtilis 168 (Y. Pereira and others, unpublished results). The structural gene csn was expressed in a degU32(Hy) mutant under the control of the inducible levansucrase leader region sacR. Thus the transcripts of the four different structural genes shared a similar 5' untranslated region encompassing the translation initiation signals and possessed 3' -independent terminators of very similar calculated free energies. In contrast, the original transcriptional context of these genes is varied, since they are either mono or polycistronic (sacC), lie on either the leading (sacB and amyE) or the lagging DNA strand (csn and sacC) and are expressed during either the exponential (sacB and csn) or stationary (amyE and sacC) growth phase.
Little is known about the mechanism of mRNA degradation in B. subtilis unlike that of Escherichia coli (Coburn & Mackie, 1999 ; Régnier & Arraiano, 2000
; Steege, 2000
). Three RNases involved in mRNA processing in B. subtilis have been identified: an endonuclease, RNase III (Wang & Bechhofer, 1997
), and two 3'5' exonucleases, polynucleotide phosphorylase and the yvaJ gene product (Wang & Bechhofer, 1996
; Bechhofer & Wang, 1998
; Oussenko & Bechhofer, 2000
). It is generally considered that most mRNAs are protected against 3' exonuclease activities by a stable secondary structure at their 3' end independently from the nucleotide sequence (Higgins et al., 1993
), and that degradation is initiated by upstream endonucleolytic cleavage (Bouvet & Belasco, 1992
) that depends mainly on 5' binding and sliding of endonucleases, although no homologue of the E. coli RNase E has yet been identified in B. subtilis (Kunst et al., 1997
; Condon et al., 1997
; Kaberdin et al., 1998
). Furthermore, transcripts of Gram-positive bacteria have been shown to contain 5' motifs that confer stability on heterologous downstream mRNAs (Bechhofer, 1993
). Such motifs are the 5' mRNA stabilizer of the erm genes of B. subtilis (Bechhofer & Dubnau, 1987
; Bechhofer & Zen, 1989
), the 5' polypurine sequence of the B. subtilis SP82 phage (Hue et al., 1995
) and the ShineDalgarno (SD)-like sequence of the cryIIIA mRNA of Bacillus thuringiensis (Agaisse & Lereclus, 1996
) which form stable complexes with ribosomes in B. subtilis. To test this potentiality further, we introduced a short sequence complementary to the end of the 16S rRNA in different places in the sacR region of the sacC transcript.
We show in this study that differences in the stabilities of the respective mRNAs may account for the differences in the production yield of the four native exoproteins. Moreover, the presence of a 5' mRNA stabilizer upstream from the RBS of sacC can lead to an increase in both the steady-state level and the stability of the mRNA and likewise to an increase in the amount of levanase released into the culture supernatant.
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METHODS |
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mRNA half-life measurements.
Rifampicin (100 µg ml-1 final concentration) was added to exponentially growing cultures at an OD600 of 1·5. Aliquots of 5 ml culture suspension were collected at intervals and immediately frozen in liquid nitrogen. RNAs were prepared as described above and mRNA half-lives were determined after Northern blotting with labelled probes as follows. RNA bands were revealed by phosphor-imaging and relative amounts of mRNA at each time quantified using ImageQuant software (Molecular Dynamics). The kinetic data of mRNA decay were analysed by non-linear least squares fitting to the sum of exponentials (SigmaPlot curve fitter program):
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where (mRNA0)i is the initial amount of species i, and i is the apparent rate constant of the species i decrease. Two models were proposed, a monophasic decay (i=1) or a biphasic decay (i=2). The model which fitted the data with the minimum deviation in each case was retained as being the more valid.
Northern blotting.
Total RNA samples (5 or 10 µg) were separated by electrophoresis on agarose gels under the conditions described by Ausubel et al. (1994) and then transferred onto a positively charged polyamide membrane (Nytran+; Schleicher & Schuell) in 20x SSC using a vacuum blotter (Hybaid). Hybridization with the probes described in Fig. 2
was carried out in a solution containing 5x SSC (1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7), 5x Denhardts solution, denatured salmon sperm DNA (100 µg ml-1 final concentration) and 1% (w/v) SDS in 50% (v/v) formamide at 42 °C, overnight. Labelling of the probes was performed either by random priming (Megaprime; Amersham) and [
-33P]dATP or with T4 polynucleotide kinase and [
-33P]dATP. In addition to the initial quantification of the total RNA by spectrophotometric measurements before electrophoresis on agarose gel, 5S rRNA, used as an internal control for standardization of the RNA samples, was probed with a 5' end-labelled oligonucleotide (Table 2
), which anneals to the 5S rRNA between nucleotides 59 and 78. Co-migration with a molecular mass marker mixture (9488363 nt RNA molecular mass markers; USB) made it possible to estimate the length of the transcripts. RNA bands were revealed by phosphor-imaging and quantified with ImageQuant software.
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sacR-directed mutagenesis.
Plasmid pGMC9, containing the sacR sequence with a minor modification (Leloup et al., 1999 ), was used to introduce a 5' mRNA stabilizer sequence by the Quick change site-directed mutagenesis method (Stratagene) with complementary oligonucleotides (Table 2
) to create, by substitution (sacRa) or insertion (sacRb and sacRc), a sequence 5'-GAAAGGAGG-3' at various positions as shown in Fig. 3
. This gave pGMC15, pGMC16 and pGMC17 containing the sacRa, sacRb and sacRc mutant fragments, respectively. The structural gene of sacC was fused to the different sacR fragments. The resulting sacRsacC fusions were controlled by sequencing, ligated into pGMK50 and used to transform strain GM96100 as described previously (Leloup et al., 1997
), giving strains GM96202, GM96203 and GM96204, respectively.
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RESULTS |
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Stability of functional mRNAs. The functional mRNA decay was evaluated by quantifying the increase in the amount of enzyme synthesized after inhibition of transcription initiation. The half-life of levansucrase functional mRNA determined previously for the same strain under the same growth conditions (Chambert & Petit-Glatron, 1984 ) was evaluated at 100±7 s. A similar approach was experimentally suitable for AmyE and SacC, but not for Csn, due to the poor sensitivity of the assay method of this enzyme (Tominaga & Tsujisaka, 1975
). Our results (Fig. 6a
, b
) show that the functional mRNA decay of sacRamyE and sacRsacC constructs fitted with monophasic events. Their half-lives were estimated to be 103±21 and 129±18 s, respectively. For
-amylase, the value of the functional half-life (103 s) was close to that of the half-life of the more stable transcript species (Fig. 5
) determined by direct measurement by Northern blotting. For levanase, the value was much higher than that of the full-length mRNA.
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DISCUSSION |
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Measurements of the steady-state level and stability of the mRNAs of the four proteins also indicated differences. Whether these differences could be caused by the minor modifications introduced in the RBS spacer regions of sacR in the various constructs should be discussed. First, the sacRamyE transcript has a shorter half-life than sacRcsn and the production of -amylase is lower than that of chitosanase, although the two transcripts possess identical spacer regions (length and base sequence). Second, in contrast, sacRsacB and sacRcsn transcripts, which show two differences (one substitution and one insertion) in their spacer region, present no significant differences in their stability, and the production of chitosanase and levansucrase is similar under the same conditions. Finally, during the revision process of the manuscript, we constructed a second sacRsacB fusion, based on the sacR fragment of plasmid pGMC9 (Leloup et al., 1999
), which was also used to construct the sacRsacC gene fusion. We observed an identical production of levansucrase by the strain harbouring this fusion, despite the two substitutions in the spacer region (data not shown). From this set of results, we concluded that the modifications introduced in the RBS spacer regions of the various constructs could not account for the discrepancies observed in the stability and in the translational efficiency of their respective mRNAs. We can now discuss other hypotheses.
In prokaryotic cells various factors are known to affect mRNA stability and therefore the steady-state level of the transcript. These factors include both gene-specific characteristics and bulk cellular conditions (Carrier & Kiesling, 1997 ), and can be divided into several categories which we will examine below.
The effects of protein synthesis on mRNA stability include translational initiation and elongation rate as well as codon usage. It has been noted that the expression of proteins can be controlled by codon usage according to the cellular levels of tRNA, which fluctuate with growth conditions and growth phase (Saier, 1995 ; Shields & Sharp, 1987
; Karlin & Mrazek, 2000
). However, the different gene fusions constructed were all expressed under the same growth conditions and during the same growth phase. Moreover, comparison of codon usage in amyE, sacC and csn showed that it is very similar for all three, but differs greatly from that of sacB (Nitschké et al., 1998
). Thus, a global effect of codon usage is highly improbable, although one should not rule out the possibility of a local effect on the rate of elongation which leaves sites unprotected from endonucleolytic attack.
Moreover, the 5' untranslated region contains a hairpin (ribonucleic antiterminator sequence) involved in the antitermination mechanism (Crutz et al., 1990 ; Aymerich & Steinmetz, 1992
) which regulates the transcription of genes under the control of sacR and can protect them, at least partially, from 5' endonuclease attack (Emory et al., 1992
; Bechhofer, 1993
). The free energies of stabilization of the four terminators at the 3' end are all very similar, therefore excluding the hypothesis that the differential stability is caused by differences in hairpins, which are supposed to prevent exonucleases from degrading the coding sequence of the mRNA (Higgins et al., 1993
). Finally, the reasons for the low stability of amyE and sacC transcripts could lie in the translation mechanism adapted to a particular growth phase (Saier, 1995
), hence the features of the translation machinery of the exponential phase did not fit well with these transcripts. The double exponential decay of amyE mRNA in addition to the monophasic decay of the corresponding functional mRNA suggests that this mRNA exists in two different states in the cells: one state with a very short half-life, probably either unprotected or only poorly protected by ribosomes, and therefore highly sensitive to endonucleolytic attack, and the other ribosome-bound with a half-life within the same range as that of sacB and csn transcripts. A similar hypothesis concerning the coexistence in the cell of functional and inactivated mRNA was proposed by Petersen (1993)
.
This hypothesis was supported by the effects of a 5' mRNA stabilizer on the stability of sacC mRNA. The effects strongly depend on the distance between the sequence and the RBS. An increase in levanase production was observed mainly in strain GM96202 (sacRasacC fusion) where the SD sequence was created by punctual mutations and was located at a distance of 34 nt from the RBS without any ATG codon between that could modify the initiation of translation. Such an increase unambiguously correlated with an increase in the steady-state level of mRNA resulting from an increase in its stability. The sacRcsacC mRNA steady-state level is slightly increased while the production of levanase is decreased. In this case, the SD sequence was introduced at a distance of 171 nt from the RBS in the very first segment of the transcript. It seems that stalling of ribosomes sterically protects the mRNA and therefore delays degradation, as found previously by Bechhofer & Dubnau (1987) . The cause of decreased protein production is not obvious. In fact the RNA-binding of SacY, the antiterminator protein, in this region (Crutz et al., 1990
) complicated our interpretation of the results obtained.
The last point that deserves discussion concerns the accumulation of an apparently homogeneous short transcript obtained with the various sacRsacC fusions. Could this be due to a premature release of the RNA polymerase or to 3'5' degradation?
Both events could result from the presence of a secondary structure in this region (Farr et al., 1999 ). The prediction obtained from the MFOLD program (Zuker et al., 1999
) shows that there is a high probability that this region contains two stemloops, the first being followed by a sequence rich in A and U (Fig. 8
). The calculated free energy of these loops is similar to that of the
-independent terminators of the four proteins. Finally, the decay of the short transcript is faster than that of the full-length transcript while the proportion of the latter is two times lower. We thus favour the hypothesis that the short transcript results from premature release of the mRNA by the polymerase rather than nuclease degradation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1994). Current Protocols in Molecular Biology. New York: Wiley Interscience.
Aymerich, S. & Steinmetz, M. (1992). Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc Natl Acad Sci USA 89, 10410-10414.[Abstract]
Bechhofer, D. H. (1993). 5' mRNA stabilizers. In Control of Messenger RNA Stability , pp. 31-52. Edited by J. G. Belasco & G. Brawerman. London:Academic Press.
Bechhofer, D. H. & Dubnau, D. (1987). Induced stability in Bacillus subtilis. Proc Natl Acad Sci USA 84, 498-502.[Abstract]
Bechhofer, D. H. & Wang, W. (1998). Decay of ermC messenger RNA in a polynucleotide phosphorylase mutant of Bacillus subtilis. J Bacteriol 180, 5968-5977.
Bechhofer, D. H. & Zen, K. (1989). Mechanism of erythromicin-induced ermC mRNA stability in Bacillus subtilis. J Bacteriol 171, 5803-5811.[Medline]
Bouvet, P. & Belasco, J. G. (1992). Control of RNase E-mediated RNA degradation by 5' termini base pairing in Escherichia coli. Nature 3, 488-491.
Carrier, T. A. & Kiesling, J. D. (1997). Controlling messenger RNA stability in bacteria: strategies for engineering gene expression. Biotechnol Prog 13, 699-708.[Medline]
Chambert, R. & Petit-Glatron, M. F. (1984). Hyperproduction of extracellular levansucrase by Bacillus subtilis: examination of the phenotype of a sacUh strain. J Gen Microbiol 130, 3143-3152.[Medline]
Chambert, R., Rain-Guion, M. C. & Petit-Glatron, M. F. (1992). Readthrough of the Bacillus subtilis levansucrase stop codon produces an extended enzyme displaying a higher polymerase activity. Biochim Biophys Acta 1132, 145-153.[Medline]
Coburn, G. A. & Mackie, G. A. (1999). Degradation of mRNA in Escherichia coli: an old problem with some new twists. Prog Nucleic Acid Res Mol Biol 62, 55-108.[Medline]
Condon, C., Putzer, H., Luo, D. & Grunberg-Manago, M. (1997). Processing of the Bacillus subtilis thrS leader mRNA is RNase E-dependent in Escherichia coli. J Mol Biol 268, 235-242.[Medline]
Crutz, A. M., Steinmetz, M., Aymerich, S., Richter, R. & Le Coq, D. (1990). Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J Bacteriol 17, 1043-1050.
Dion, M., Rapoport, G. & Doly, J. (1989). Expression of the MuIFN7 gene in Bacillus subtilis using the levansucrase system. Biochimie 71, 747-755.[Medline]
Emory, S. A., Bouvet, P. & Belasco, J. G. (1992). A 5'-terminal stemloop structure can stabilize mRNA in Escherichia coli. Genes Dev 6, 135-148.[Abstract]
Farr, G. A., Oussenko, I. A. & Bechhofer, D. H. (1999). Protection against 3' to 5' RNA decay in Bacillus subtilis. J Bacteriol 181, 7323-7330.
Higgins, C. F., Causton, H. C., Dance, G. S. C. & Mudd, E. A. (1993). The role of the 3' end in mRNA stability and decay. In Control of Messenger RNA Stability , pp. 13-30. Edited by J. G. Belasco & G. Brawerman. London:Academic Press.
Horionouchi, S. & Weisblum, B. (1982). Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150, 815-825.[Medline]
Hue, K. K., Cohen, S. D. & Bechhofer, D. H. (1995). A polypurine sequence that acts as a 5' stabilizer in Bacillus subtilis. J Bacteriol 177, 3465-3471.[Abstract]
Joliff, G., Edelman, A., Klier, A. & Rapoport, G. (1989). Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl Environ Microbiol 55, 2739-2744.
Joyet, P., Levin, D., de Louvencourt, L., Le Révérent, B., Aymerich, A. & Heslot, H. (1986). Expression of a thermostable alpha-amylase gene under the control of levansucrase inducible promoter from Bacillus subtilis. In Bacillus Molecular Genetics and Biotechnology Applications , pp. 470-491. Edited by A. T. Ganesan & J. A. Hoch. London:Academic Press.
Kaberdin, V. R., Miczak, A., Jakobsen, J. S., Lin-Chao, S., McDowall, K. J. & von Gabain, A. (1998). The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly. Proc Natl Acad Sci USA 95, 11637-11642.
Karlin, S. & Mrazek, J. (2000). Predicted highly expressed genes of diverse prokaryotes. J Bacteriol 182, 5238-5250.
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Leloup, L., Haddaoui, E., Chambert, R. & Petit-Glatron, M. F. (1997). Characterization of the rate limiting step of the secretion of Bacillus subtilis -amylase overproduced during the exponential phase of growth. Microbiology 143, 3295-3303.[Abstract]
Leloup, L., Le Saux, J., Petit-Glatron, M. F. & Chambert, R. (1999). Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth. Microbiology 145, 613-619.[Abstract]
Lepesant, J. A., Kunst, F., Pascal, J., Kejzlarova-Lepesant, Steimetz, M. & Dedonder, R. (1976). Specific and pleiotropic regulatory mechanisms in the sucrose system of Bacillus subtilis 168. In Microbiology 1976, pp. 5869. Edited by D. Schlessinger. Washington, DC: American Society for Microbiology.
Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1990). Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol 214, 657-671.[Medline]
Masson, J. Y., Denis, F. & Brezinski, R. (1994). Primary sequence of the chitosanase from Streptomyces sp. strain N174 and comparison with other endoglycosidases. Gene 140, 103-107.[Medline]
Nitschké, P., Guerdoux-Jamet, P., Chiapello, H., Faroux, G., Henaut, C., Henaut, A. & Danchin, A. (1998). Indigo: a World-Wide-Web review of genomes and gene functions. FEMS Microbiol Rev 22, 207-227.[Medline]
Oussenko, I. A. & Bechhofer, D. H. (2000). The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase. J Bacteriol 182, 2639-2642.
Parro, V., San Roman, M., Galindo, I., Purnelle, B., Bolotin, A., Sorokin, A. & Mellado, R. P. (1997). A 23911 nucleotide region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon. Microbiology 143, 1321-1326.[Abstract]
Petersen, C. (1993). Translation and mRNA stability in bacteria: a complex relationship. In Control of Messenger RNA Stability , pp. 117-145. Edited by J. G. Belasco & G. Brawerman. London:Academic Press.
Petit, M. A., Joliff, G., Mesas, J. M., Klier, A., Rapoport, G. & Ehrlich, S. D. (1990). Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification. Biotechnology 8, 559-563.[Medline]
Petit-Glatron, M. F. & Chambert, R. (1992). Peptide carrier potentiality of Bacillus subtilis levansucrase. J Gen Microbiol 138, 1089-1095.[Medline]
Priest, F. G. (1977). Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev 41, 711-753.[Medline]
Putzer, H., Gendron, N. & Grunberg-Manago, M. (1992). Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence. EMBO J 11, 3117-3127.[Abstract]
Régnier, P. & Arraiano, C. M. (2000). Degradation of mRNA in bacteria: emergence of ubiquitous features. Bioessays 22, 235-244.[Medline]
Saier, M. H.Jr (1995). Differential codon usage: a safeguard against inappropriate expression of specialized genes? FEBS Lett 362, 1-4.[Medline]
Shields, D. C. & Sharp, P. M. (1987). Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Res 15, 8023-8040.[Abstract]
Shimotsu, H. & Henner, D. J. (1986). Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of steady-state mRNA level by sacU and sacQ genes. J Bacteriol 168, 380-388.[Medline]
Steege, D. A. (2000). Emerging features of mRNA decay in bacteria. RNA 6, 1079-1090.
Steinmetz, M., Le Coq, D., Aymerich, S., Gonzy-Tréboul, G. & Gay, P. (1985). The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet 200, 220-228.[Medline]
Tominaga, Y. & Tsujisaka, Y. (1975). Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls. Biochim Biophys Acta 410, 145-155.[Medline]
Wang, W. & Bechhofer, D. H. (1996). Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain. J Bacteriol 178, 2375-2382.[Abstract]
Wang, W. & Bechhofer, D. H. (1997). Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci. J Bacteriol 179, 7379-7385.[Abstract]
Wong, S. L. (1989). Development of an inducible and enhancible expression and secretion system in Bacillus subtilis. Gene 83, 215-223.[Medline]
Yamaguchi, K., Nagata, Y. & Maruo, B. (1974). Genetic control of the rate of alpha amylase synthesis in Bacillus subtilis. J Bacteriol 119, 410-415.[Medline]
Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction. A practical guide. In RNA Biochemistry and Biotechnology. Edited by J. Barciszewski & B. F. C. Clark. NATO ASI Series: Kluwer Academic Publishers.
Received 14 November 2000;
revised 22 January 2001;
accepted 8 February 2001.