Elongation and Proofreading in E. Coli / E. Coli Transcription Regulation and Operons

Pausing and backtracking

Polymerase can pause/backtrack to coordinate with translation and resolve issues

Hydrolytic proofreading

GreA/GreB stimulate cleavage to remove misincorporated nucleotides and resume elongation

Intrinsic termination logic

RNA hairpin formation plus an AU-rich region destabilizes the RNA-DNA hybrid to release RNA

Rho-dependent termination

Rho binds rut sites and uses ATP to catch polymerase and unwind RNA-DNA heteroduplex

Housekeeping vs conditional genes

some genes are constitutive, while others respond to environmental signals.

Negative control mechanism

Repressors bind operators to block polymerase binding/initiating near the +1 site

Positive control mechanism

Activators increase the frequency of productive polymerase-promoter interactions

cis-regulatory definition

Promoters/operators are DNA elements that act on linked genes

Operon definition

multiple genes share regulatory DNA and produce a polycistronic mRNA

Operon advantages

coordinated expression yields matched protein quantities with fewer regulatory elements

Lac operon state logic

Glucose and lactose availability controls CAP activity and Lac repressor binding to tune lac transcription output

Reporter assay purpose

Fuse promoter/regulatory DNA to a reporter gene to quantify promoter strength by reporter output

Up vs Down mutations

“Up” mutations move sequences toward consensus to increase expression, while “down” mutations reduce expression

Linker scanning idea

Replace contiguous promoter segments with neutral linker DNA to locate functional regions