TY - JOUR
T1 - Nascent RNA in transcription complexes interacts with CspE, a small protein in E. coli implicated in chromatin condensation
AU - Hanna, Michelle M.
AU - Liu, Kebin
N1 - Funding Information:
This work was support by NIH grant RO1 GM47493 and NSF grant MCB-9509132 to M.M.H. We thank Nancy Trun for discussions and sharing unpublished data regarding the function of CspE. We are grateful to the Molecular Biology Facility at the University of Oklahoma Health Sciences Center for protein sequencing and mass spectrometry analysis.
PY - 1998/9/18
Y1 - 1998/9/18
N2 - Proteins in a partially fractionated Escherichia coli extract that interact with the nascent RNA in active transcription complexes from several promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as transcription substrates. Upon irradiation of ternary transcription complexes, several extract proteins were crosslinked to the RNA. Most notably, a small protein was crosslinked to the RNA in complexes on seven of nine templates tested. This protein was purified and sequenced and found to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning. CspE has 69% amino acid sequence identity with the major cold shock protein in E. coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the sequence 5'-ATTGG. Of the nine templates tested, CspE was found to be most heavily crosslinked to RNA from the λ P(R) promoter, which is modified by the Q antiterminator protein. CspE was very heavily crosslinked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released from the transcription complex. However, even when present from the start of transcription, CspE did not crosslink to the RNA 82 nucleotides long in elongation complexes from this same promoter. Despite the loss of interaction with the RNA after polymerase had left the promoter, CspE inhibited Q-mediated transcriptional antitermination from P(R), in vitro almost 200 nucleotides downstream from the promoter, presumably by interaction with the Y box DNA upstream from P(R), which overlaps with the binding site for the Q. A potential role for CspE and transcription in chromosome condensation and nucleoid structure is discussed.
AB - Proteins in a partially fractionated Escherichia coli extract that interact with the nascent RNA in active transcription complexes from several promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as transcription substrates. Upon irradiation of ternary transcription complexes, several extract proteins were crosslinked to the RNA. Most notably, a small protein was crosslinked to the RNA in complexes on seven of nine templates tested. This protein was purified and sequenced and found to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning. CspE has 69% amino acid sequence identity with the major cold shock protein in E. coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the sequence 5'-ATTGG. Of the nine templates tested, CspE was found to be most heavily crosslinked to RNA from the λ P(R) promoter, which is modified by the Q antiterminator protein. CspE was very heavily crosslinked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released from the transcription complex. However, even when present from the start of transcription, CspE did not crosslink to the RNA 82 nucleotides long in elongation complexes from this same promoter. Despite the loss of interaction with the RNA after polymerase had left the promoter, CspE inhibited Q-mediated transcriptional antitermination from P(R), in vitro almost 200 nucleotides downstream from the promoter, presumably by interaction with the Y box DNA upstream from P(R), which overlaps with the binding site for the Q. A potential role for CspE and transcription in chromosome condensation and nucleoid structure is discussed.
KW - CspE
KW - Photocrosslinking nucleotide analogs
KW - Q
KW - RNA and DNA binding protein
KW - Transcriptional antitermination
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U2 - 10.1006/jmbi.1998.2005
DO - 10.1006/jmbi.1998.2005
M3 - Article
C2 - 9735283
AN - SCOPUS:0032544629
SN - 0022-2836
VL - 282
SP - 227
EP - 239
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -