Characterization of the Interactions between the Bacteriophage T4 AsiA Protein and RNA Polymerase
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- 作者：Mario F. Simeonov ; Ramona J. Bieber Urbauer ; Joshua M. Gilmore ; Karen Adelman ; Edward N. Brody ; Anita Niedziela-Majka ; Leonid Minakhin ; Tomasz Heyduk ; and Jeffrey L. Urbauer
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：July 1, 2003
- 年：2003
- 卷：42
- 期：25
- 页码：7717 - 7726
- 全文大小：375K
- 年卷期：v.42,no.25(July 1, 2003)
- ISSN：1520-4995
文摘
The anti-
factor AsiA effects a change in promoter specificity of the Escherichia coli RNApolymerase via interactions with two conserved regions of the 70 subunit, denoted 4.1 and 4.2. FreeAsiA is a symmetrical homodimer. Here, we show that AsiA is monomeric when bound to 70 and thata subset of the residues that contribute to the homodimer interface also contributes to the interface with70. AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkablesequence similarity. An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolatedregions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interactwith either subsite. These results suggest structural and functional plasticity in the interaction of AsiAwith 70 and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation byAsiA. Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element byregion 4 of 70 indirectly, as the residues on region 4 responsible for AsiA binding are distinct from thoseinvolved in DNA binding. Finally, we show that AsiA must directly disrupt the interaction of region 4with the RNA polymerase 
subunit flap domain, resulting in a distance change between region 2 andregion 4 of 70. Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby thedistance between the DNA binding domains in 70 is regulated, and promoter recognition specificity ismodulated, by mediating the interactions of the region 4 with the subunit flap domain.
factor AsiA effects a change in promoter specificity of the Escherichia coli RNApolymerase via interactions with two conserved regions of the 70 subunit, denoted 4.1 and 4.2. FreeAsiA is a symmetrical homodimer. Here, we show that AsiA is monomeric when bound to 70 and thata subset of the residues that contribute to the homodimer interface also contributes to the interface with70. AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkablesequence similarity. An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolatedregions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interactwith either subsite. These results suggest structural and functional plasticity in the interaction of AsiAwith 70 and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation byAsiA. Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element byregion 4 of 70 indirectly, as the residues on region 4 responsible for AsiA binding are distinct from thoseinvolved in DNA binding. Finally, we show that AsiA must directly disrupt the interaction of region 4with the RNA polymerase subunit flap domain, resulting in a distance change between region 2 andregion 4 of 70. Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby thedistance between the DNA binding domains in 70 is regulated, and promoter recognition specificity ismodulated, by mediating the interactions of the region 4 with the subunit flap domain.