Structure of a transcribing T7 RNA polymerase initiation complex

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• T7 RNA polymerase (T7 RNAP) is a crucial enzyme for gene expression in various biological systems.
• Understanding the initiation phase of transcription is vital for controlling gene expression.
• Previous studies have characterized T7 RNAP's open promoter complex structure.

Purpose of the Study:

• To elucidate the structural basis of T7 RNA polymerase during the initial phase of RNA synthesis.
• To investigate the mechanism of template repositioning and RNA accumulation at the active site.

Main Methods:

• Determined the high-resolution (2.4 angstroms) crystal structure of the T7 RNAP-promoter DNA-trinucleotide RNA complex.
• Analyzed the interaction between the promoter DNA, single-stranded template extension, and the T7 RNAP active site.

Main Results:

• The upstream duplex promoter DNA binds similarly to the open complex.
• The single-stranded template is repositioned, placing the +4 base at the catalytic active site.
• RNA synthesis causes template accumulation ('scrunching') within the confined active site pocket, limiting heteroduplex formation to three base pairs before RNA release.

Conclusions:

• The structure reveals a novel template scrunching mechanism during T7 RNAP initiation.
• This mechanism facilitates efficient RNA synthesis initiation while limiting promoter melting.
• The findings provide insights into the regulation of transcription initiation by viral polymerases.