RNA polymerase from Rhizobium japonicum

Area of Science:

• Molecular Biology
• Enzymology
• Microbiology

Background:

• DNA-dependent RNA polymerase is crucial for gene expression in bacteria.
• Rhizobium japonicum is a nitrogen-fixing bacterium with unique metabolic pathways.

Purpose of the Study:

• To purify and characterize the DNA-dependent RNA polymerase from Rhizobium japonicum.
• To investigate potential modifications of the enzyme during nitrogen fixation.

Main Methods:

• Enzyme purification using electrophoresis.
• Determination of subunit structure and molecular weights.
• Assessing enzyme activity and promoter binding.

Main Results:

• The subunit structure was determined as beta beta' alpha 2 sigma.
• Apparent molecular weights for subunits and holoenzyme were established.
• No electrophoretic differences were observed between enzymes from aerobic and nitrogen-fixing cells.
• The enzyme demonstrated Mg2+-dependence, rifampicin-sensitivity, and optimal activity at alkaline pH and 35-40°C.
• Differential promoter binding was observed, with strong binding to T7 promoters and no binding to R. japonicum nif DNA.

Conclusions:

• The purified RNA polymerase from R. japonicum is a multi-subunit enzyme with a defined structure.
• Metabolic differentiation into nitrogen-fixing cells does not involve chemical modification of RNA polymerase.
• Additional factors are likely required for the selective transcription of nif genes in R. japonicum.