Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases.
Transcription ternary complexes of Escherichia coli RNA polymerase and yeast RNA polymerase III have been analyzed by atomic force microscopy. Using the method of nucleotide omission and different DNA templates, E.coli RNAP has been stalled at position +24, +70 and +379 and RNAP III at position +377 from the starting site. Conformational analysis of E.coli RNAP elongation complexes reveals an average DNA compaction of 22nm and a DNA deformation compatible with approximately 180 degrees DNA wrapping against the enzyme. The extent of protein-DNA interaction attributed to wrapping, however, is less than that of corresponding open promoter complexes. DNA wrapping was also observed for RNAP III elongation complexes, which showed a DNA compaction of 30nm. When the RNA polymerases were stalled far from the promoter (+379 and +377), the growing RNA transcript was often visible and it was prevalently seen exiting from the enzyme on the opposite side relative to the smallest angle subtended by the upstream and downstream DNA arms. Surprisingly, we found that many complexes had a second RNAP, not involved in transcription, bound to the growing RNA of a ternary complex. DNA wrapping in the elongation complex suggests a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.
Rivetti
Claudio
C
Department of Biochemistry and Molecular Biology, University of Parma, 43100, Parma, Italy. rivetti@unipr.it
Codeluppi
Simone
S
Dieci
Giorgio
G
Bustamante
Carlos
C
D001426:Bacterial Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009707:Nucleosomes; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III