Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation

Area of Science:

• Molecular Biology
• Enzymology
• Genetics

Background:

• The beta subunit of Escherichia coli RNA polymerase contains an evolutionarily conserved segment (Asp1064-Lys1073) near the nucleotide binding pocket.
• Previous studies used affinity labeling to map this region's proximity to priming substrates.

Purpose of the Study:

• To investigate the functional importance of conserved amino acids in the Asp1064-Lys1073 segment of E. coli RNA polymerase.
• To determine the effects of specific amino acid substitutions on RNA polymerase activity, initiation, and elongation.

Main Methods:

• Site-directed mutagenesis was used to create single amino acid substitutions (Xaa-->Ala or Ala-->Ser) and a multiple alanine substitution (KRNK) in the target segment.
• Mutant RNA polymerases were prepared via in vitro reconstitution.
• Biochemical assays were performed to assess promoter complex formation, RNA synthesis initiation, abortive initiation, promoter clearance, and elongation pausing.

Main Results:

• Mutations ranged in severity from viable to dominant or recessive lethal.
• All viable mutant enzymes formed stable promoter complexes and initiated RNA synthesis.
• The KRNK mutant was blocked in the initiation-to-elongation transition.
• Point mutants showed allele-specific alterations in promoter clearance rates, abortive initiation patterns, and elongation pausing.

Conclusions:

• The studied mutations distort but do not destroy the RNA polymerase active center.
• Specific amino acid substitutions in this conserved segment alter the coupling between catalysis and DNA translocation.
• These findings provide insights into the mechanism of RNA polymerase function and regulation.