Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis.

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.

[1]  H. Domdey,et al.  Nucleotide sequence and secondary structure of potato spindle tuber viroid , 1978, Nature.

[2]  B. Ding,et al.  Cell-to-cell movement of potato spindle tuber viroid. , 1997, The Plant journal : for cell and molecular biology.

[3]  D Riesner,et al.  Multiple pathways of reversion in viroids for conservation of structural elements. , 1993, The EMBO journal.

[4]  G. Steger,et al.  Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing. , 1988, Gene.

[5]  R Nussinov,et al.  Sequential folding of a messenger RNA molecule. , 1981, Journal of molecular biology.

[6]  D Riesner,et al.  Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. , 1991, The EMBO journal.

[7]  J. Remme,et al.  Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase. , 1993, Journal of molecular biology.

[8]  S. Kim,et al.  Sequential folding of transfer RNA. A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5' end. , 1980, Journal of molecular biology.

[9]  H. L. Sänger,et al.  Viroid replication is inhibited by α-amanitin , 1979, Nature.

[10]  K. Henco,et al.  Conformation of viroids , 1977, Nucleic Acids Res..

[11]  C Frieden,et al.  New PC versions of the kinetic-simulation and fitting programs, KINSIM and FITSIM. , 1997, Trends in biochemical sciences.

[12]  M. A. Rezaian,et al.  A scheme for viroid classification. , 1989, Intervirology.

[13]  D. Riesner,et al.  Fine structure melting of viroids as studied by kinetic methods. , 1979, Nucleic acids research.

[14]  T. Baumstark,et al.  Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract. , 1995, Nucleic acids research.

[15]  D Riesner,et al.  Temperature‐Gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations, and protein‐nucleic acid interactions , 1989, Electrophoresis.

[16]  D. Riesner,et al.  Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts. , 1987, Biophysical chemistry.

[17]  Detlev Riesner,et al.  Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation , 1997, The EMBO journal.

[18]  S. Lin-Chao,et al.  RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColE1-type plasmids in Escherichia coli. , 1995, Nucleic acids research.

[19]  M. Chamberlin,et al.  Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template. , 1982, The Journal of biological chemistry.

[20]  G. Steger,et al.  Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data. , 1984, Journal of biomolecular structure & dynamics.

[21]  H. Masukata,et al.  A mechanism of formation of a persistent hybrid between elongating RNA and template DNA , 1990, Cell.

[22]  J. Randles,et al.  A proposed scheme for viroid classification and nomenclature , 1998, Archives of Virology.

[23]  M. Chamberlin,et al.  Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis. , 1974, The Journal of biological chemistry.

[24]  Oligomeric potato spindle tuber viroid (PSTV) RNA does not process autocatalytically under conditions where other RNAs do. , 1987, Virology.

[25]  Ines Schindler,et al.  Involvement of nuclear DNA-dependent RNA polymerases in potato spindle tuber viroid replication: a reevaluation , 1992 .

[26]  M. Chamberlin,et al.  Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. , 1973, The Journal of biological chemistry.

[27]  C Frieden,et al.  Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. , 1983, Analytical biochemistry.

[28]  D. Riesner,et al.  A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. , 1983, Analytical biochemistry.

[29]  D. Riesner,et al.  Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy. , 1989, The EMBO journal.