Related Experiment Videos
Probing yeast RNA polymerase A subunits with monospecific antibodies
Abstract:
Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
Insights
Researchers developed monoclonal antibodies targeting specific subunits of yeast RNA polymerase A. These antibodies are valuable tools for investigating RNA polymerase subunit function in transcription.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- RNA polymerase A is crucial for transcription in Saccharomyces cerevisiae.
- Understanding the function of individual subunits is key to elucidating transcription mechanisms.
Purpose of the Study:
- To generate and characterize monoclonal antibodies against specific subunits of yeast RNA polymerase A.
- To investigate the role of different subunits in RNA polymerase activity and function.
Main Methods:
- Monoclonal antibody production in mice against native and dissociated RNA polymerase A.
- Screening using spot-immunodetection and specificity analysis via blot-immunodetection.
- In vitro transcription assays and preincubation with DNA to assess antibody effects.
Main Results:
- 14 hybridomas producing antibodies against RNA polymerase A subunits A135, A49, A43, and A14.5 were selected.
- An antibody against subunit A190 was also generated.
- Antibodies against A135 inhibited in vitro RNA synthesis, suggesting a role in DNA binding.
- An antibody against A14.5 recognized a common subunit across yeast nuclear RNA polymerases without affecting activity.
Conclusions:
- Monoclonal antibodies against specific RNA polymerase subunits have been successfully generated.
- The A135 subunit is implicated in DNA template binding during transcription.
- These antibody probes offer a valuable resource for studying subunit function in reconstituted transcription systems.