RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes.

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.

[1]  M. Chamberlin,et al.  Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites. , 1989, Journal of molecular biology.

[2]  J. Kahn,et al.  Reversibility of nucleotide incorporation by Escherichia coli RNA polymerase, and its effect on fidelity. , 1989, Journal of molecular biology.

[3]  R. Saiki,et al.  A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. , 1988, Nucleic acids research.

[4]  Donald M. Crothers,et al.  Lac repressor is a transient gene-activating protein , 1987, Cell.

[5]  D. Luse,et al.  Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter. , 1987, The Journal of biological chemistry.

[6]  M. Chamberlin,et al.  Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at a single template base. , 1987, Journal of molecular biology.

[7]  D. Dennis,et al.  RNA polymerase. Limit cognate primer for initiation and stable ternary complex formation. , 1987, The Journal of biological chemistry.

[8]  D. Crothers,et al.  A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. , 1987, Journal of molecular biology.

[9]  H. Bujard,et al.  Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. , 1986, The EMBO journal.

[10]  H. Bujard,et al.  Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. , 1986, The EMBO journal.

[11]  E. Geiduschek,et al.  Transcription at bacteriophage T4 variant late promoters. An application of a newly devised promoter-mapping method involving RNA chain retraction. , 1986, The Journal of biological chemistry.

[12]  C. Meares,et al.  The sigma subunit of RNA polymerase contacts the leading ends of transcripts 9-13 bases long on the lambda PR promoter but not on T7 A1. , 1986, Biochemistry.

[13]  N. Shimamoto,et al.  Release of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA. , 1986, The Journal of biological chemistry.

[14]  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.

[15]  G. Hartmann,et al.  Synthesis of dinucleoside tetraphosphates by RNA polymerase B (II) from calf thymus , 1985, FEBS letters.

[16]  Donald M. Crothers,et al.  Intermediates in transcription initiation from the E. coli lac UV5 promoter , 1985, Cell.

[17]  A. Schäffner,et al.  Primer-independent abortive initiation by wheat-germ RNA polymerase B (II). , 1985, European journal of biochemistry.

[18]  J. Gralla,et al.  Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation. , 1985, Journal of molecular biology.

[19]  D. Dennis,et al.  RNA polymerase. Direct evidence for two active sites involved in transcription. , 1985, The Journal of biological chemistry.

[20]  W. McClure,et al.  Mechanism and control of transcription initiation in prokaryotes. , 1985, Annual review of biochemistry.

[21]  J. Brosius,et al.  Regulation of ribosomal RNA promoters with a synthetic lac operator. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. V. von Hippel,et al.  Protein-nucleic acid interactions in transcription: a molecular analysis. , 1984, Annual review of biochemistry.

[23]  C. Hsu,et al.  RNA polymerase: correlation between transcript length, abortive product synthesis, and formation of a stable ternary complex. , 1982, Biochemistry.

[24]  A. Chenchik,et al.  Processive pyrophosphorolysis of RNA by Escherichia coli RNA polymerase , 1982, FEBS letters.

[25]  W. Zillig,et al.  Rifampicin inhibition of RNA synthesis by destabilisation of DNA-RNA polymerase-oligonucleotide-complexes. , 1981, Nucleic acids research.

[26]  F. Studier,et al.  Genetic and physical mapping of the late region of bacteriophage T7 DNA by use of cloned fragments of T7 DNA. , 1981, Journal of molecular biology.

[27]  W. Reznikoff,et al.  Abortive initiation and long ribonucleic acid synthesis. , 1981, Biochemistry.

[28]  J. Gralla,et al.  Productive and abortive initiation of transcription in vitro at the lac UV5 promoter. , 1980, Biochemistry.

[29]  W R McClure,et al.  Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. II. Release of sigma from ternary complexes. , 1980, The Journal of biological chemistry.

[30]  J. Gralla,et al.  Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. , 1980, Biochemistry.

[31]  E. Zaychikov,et al.  Initiation by Escherichia coli RNA‐polymerase: transformation of abortive to productive complex , 1980, FEBS letters.

[32]  Walter Gilbert,et al.  E. coli RNA polymerase interacts homologously with two different promoters , 1980, Cell.

[33]  M. Chamberlin,et al.  A quantitative assay for bacterial RNA polymerases. , 1979, The Journal of biological chemistry.

[34]  ULRICH SIEBENLIST,et al.  RNA polymerase unwinds an 11-base pair segment of a phage T7 promoter , 1979, Nature.

[35]  T. Taniguchi,et al.  Unusual location and function of the operator in the Escherichia coli galactose operon , 1979, Nature.

[36]  S. Adhya,et al.  Modulation of the two promoters of the galactose operon of Escherichia coli , 1979, Nature.

[37]  M. Chamberlin,et al.  A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase. , 1977, Archives of biochemistry and biophysics.

[38]  W. Gilbert,et al.  A new method for sequencing DNA. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M J Chamberlin,et al.  The selectivity of transcription. , 1974, Annual review of biochemistry.

[40]  J. Hurwitz,et al.  The role of deoxyribonucleic acid in ribonucleic acid synthesis. 13. Modified purification procedure and additional properties of ribonucleic acid polymerase from Escherichia coli W. , 1967, The Journal of biological chemistry.

[41]  R. Shapiro,et al.  The deamination of cytidine and cytosine by acidic buffer solutions. Mutagenic implications. , 1966, Biochemistry.

[42]  J. Josse,et al.  Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of nearest neighbor base sequences in deoxyribonucleic acid. , 1961, The Journal of biological chemistry.