Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase.

The various kinetic and thermodynamic models for transcription elongation all require an understanding of the nature of the melted bubble which moves with the RNA polymerase active site. Is the general nature of the bubble system-dependent or are there common energetic requirements which constrain a bubble in any RNA polymerases? T7 RNA polymerase is one of the simplest RNA polymerases and is the system for which we have the highest-resolution structural information. However, there is no high-resolution information available for a stable elongation complex. In order to directly map melted regions of the DNA in a functionally paused elongation complex, we have introduced fluorescent probes site-specifically into the DNA. Like 2-aminopurine, which substitutes for adenine bases, the fluorescence intensity of the new probe, pyrrolo-dC, which substitutes for cytosine bases, is sensitive to its environment. Specifically, the fluorescence is quenched in duplex DNA relative to its fluorescence in single-stranded DNA, such that the probe provides direct information on local melting of the DNA. Placement of this new probe at specific positions in the non-template strand shows clearly that the elongation bubble extends about eight bases upstream of the pause site, while 2-aminopurine probes show that the elongation bubble extends only about one nucleotide downstream of the last base incorporated. The positioning of the active site very close to the downstream edge of the bubble is consistent with previous studies and with similar studies of the promoter-bound, pre-initiation complex. The results show clearly that the RNA:DNA hybrid can be no more than eight nucleotides in length, and characterization of different paused species suggests preliminarily that these dimensions are not sequence or position dependent. Finally, the results confirm that the ternary complex is not stable with short lengths of transcript, but persists for a substantial time when paused in the middle or at the (runoff) end of duplex DNA.

[1]  W. Mcallister,et al.  The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Coleman,et al.  T7 ribonucleic acid polymerase-promotor interactions. , 1981, Biochemistry.

[3]  J. Sullivan,et al.  Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase. , 1997, Biochemistry.

[4]  C. Martin,et al.  Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347. , 1986, Biochemistry.

[5]  R. Burgess,et al.  Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II). , 1987, Biochemistry.

[6]  W. Mcallister,et al.  Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. , 2000, Journal of molecular biology.

[7]  R. Sousa,et al.  T7 RNA polymerase elongation complex structure and movement. , 2000, Journal of molecular biology.

[8]  K. Evans,et al.  Melting and premelting transitions of an oligomer measured by DNA base fluorescence and absorption. , 1994, Biochemistry.

[9]  A Kumar,et al.  Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes* , 1996, The Journal of Biological Chemistry.

[10]  T. Steitz,et al.  Structure of a transcribing T7 RNA polymerase initiation complex. , 1999, Science.

[11]  E. Nudler,et al.  The RNA–DNA Hybrid Maintains the Register of Transcription by Preventing Backtracking of RNA Polymerase , 1997, Cell.

[12]  J. Hearst,et al.  RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay. , 1991, Biochemistry.

[13]  T. Steitz,et al.  Structural basis for initiation of transcription from an RNA polymerase–promoter complex , 1999, Nature.

[14]  P. V. von Hippel,et al.  Determinants of the stability of transcription elongation complexes: interactions of the nascent RNA with the DNA template and the RNA polymerase. , 1999, Journal of molecular biology.

[15]  P. Dehaseth,et al.  Protein-nucleic acid interactions during open complex formation investigated by systematic alteration of the protein and DNA binding partners. , 1999, Biochemistry.

[16]  C. Martin,et al.  Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase. , 1997, Journal of molecular biology.

[17]  W. Mcallister,et al.  Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement. , 1999, Journal of molecular biology.

[18]  C. Richardson,et al.  Interactions of a proteolytically nicked RNA polymerase of bacteriophage T7 with its promoter. , 1987, The Journal of biological chemistry.

[19]  M. Chamberlin,et al.  Structural analysis of ternary complexes of Escherichia coli RNA polymerase. Deoxyribonuclease I footprinting of defined complexes. , 1992, Journal of molecular biology.

[20]  E. Nudler,et al.  Spatial organization of transcription elongation complex in Escherichia coli. , 1998, Science.

[21]  D. Xu,et al.  Sequence dependence of energy transfer in DNA oligonucleotides. , 2000, Biophysical journal.

[22]  S. Sastry,et al.  A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase. , 1996, Biochemistry.

[23]  C. Martin,et al.  Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase. , 1996, Biochemistry.

[24]  R. Burgess,et al.  Bacteriophage T7 late promoters with point mutations: quantitative footprinting and in vivo expression. , 1988, Nucleic acids research.

[25]  R. Sousa,et al.  A model for the mechanism of polymerase translocation. , 1997, Journal of molecular biology.

[26]  C. Martin,et al.  Identification of specific contacts in T3 RNA polymerase-promoter interactions: kinetic analysis using small synthetic promoters. , 1993, Biochemistry.

[27]  R. Sousa,et al.  Structural and mechanistic relationships between nucleic acid polymerases. , 1996, Trends in biochemical sciences.

[28]  Craig T Martin,et al.  Thermodynamic and Kinetic Measurements of Promoter Binding by T 7 RNA Polymerase † , 2022 .

[29]  M. Chamberlin,et al.  Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes. , 1999, Biochemistry.

[30]  F. Studier,et al.  Cloning and expression of the gene for bacteriophage T7 RNA polymerase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Chamberlin,et al.  Basic mechanisms of transcript elongation and its regulation. , 1997, Annual review of biochemistry.

[32]  W. Mcallister,et al.  Promoter specificity determinants of T7 RNA polymerase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[33]  C. Richardson,et al.  Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. , 1986, Proceedings of the National Academy of Sciences of the United States of America.