Primers with significant secondary structure are undesirable.

#Id: 2443

#Unit 3. Fundamental Processes

The need for rho factor in vitro is variable. Some (rho-dependent) terminators require relatively high concentrations of rho, whereas others function just as well at lower levels. 

#Id: 2444

#Unit 3. Fundamental Processes

Different terminators require different levels of rho factor for termination and therefore respond differently to the residual levels of rho factor in the mutants (rho mutants are usually leaky).

#Id: 2445

#Unit 3. Fundamental Processes

The b subunit of RNA polymerase is implicated by two types of mutation. 

First, mutations in the rpoB gene can reduce termination at a rho-dependent site. 

Second, mutations in rpoB can restore the ability to terminate transcription at rho-dependent sites in rho mutant bacteria.

#Id: 2446

#Unit 3. Fundamental Processes

One consequence of the rotation of DNA is illustrated In the two-domain model for transcription, as RNA polymerase moves with respect to the double helix, it generates positive supercoils (more tightly wound DNA) ahead of it and leaves negative supercoils (partially unwound DNA) behind it. For each helical turn traversed by RNA polymerase, + 1 turn is generated ahead and -1 turn behind.

#Id: 2447

#Unit 3. Fundamental Processes

The enzymes DNA gyrase, which introduces negative supercoils into DNA, and DNA topoisomerase I, which removes negative supercoils in DNA, are required to prevent topological stresses from building up in the course of transcription and replication. 

#Id: 2448

#Unit 3. Fundamental Processes

Blocking the activities of gyrase and topoisomerase therefore result in major changes in DNA supercoiling, which in turn affects transcription and replication.