Lecture 1: Key Concepts

Question 1

What techniques can we use?

Answer 1

• We can use genetics to understand operons.
• We can also look at the effect of mutations.
• We can look at the structures of transcription factors and RNA polymerases to get insight into mechanism.
• We can also see how small molecules like cAMP affect function.
• Inhibitors can also help us to understand functions and mechanisms.
• Single molecule approaches are also useful.

Question 2

What are the three main methods of gene regulation in bacteria?

Answer 2

• DNA topology, gene context and architectural proteins.
• Non-coding RNA.
• Transcription factors.
We can integrate multiple signals as a way to affect gene transcription.

Question 3

How do DNA topology, gene context and architectural proteins affect transcription?

Answer 3

• The bacterial chromosome is negatively supercoiled. Transcription alters supercoiling. New positive supercoils can relax the DNA.
• ATP-dependent topoisomerases can be used to alter DNA supercoiling and affect DNA binding proteins.
• Changes in super helicity can alter the relative positions of -35 and -10 regions at bacterial promoters.
• DNA gyrase can generate negative supercoils into relaxed DNA and relax positively supercoiled DNA.
• Nucleoid associated proteins can change the structure of DNA in the nucleoid (region where most DNA is).
• A major example of a nucleoid associated protein is histone-like nucleoid-structuring (H-NS) protein. H-NS forms complexes with itself and binds to different sections of DNA and bring them together. H-NS binds to AT rich DNA, and it can exclude RNA polymerase from a promoter. Another protein binding can relieve this suppression.

Question 4

How do non-coding RNAs affect transcription?

Answer 4

Non-coding RNAs can include antisense transcripts as well as ribosensors and riboswitches.
• Ribosensors/riboswitches are elements present in the 5’-UTRs of mRNA molecules that may bind to ligands. They regulate the expression of downstream genes, often by controlling transcription termination or translation.
• A riboswitch is a ribozyme that can cleave itself at sufficient metabolite concentrations.
• Antisense RNAs often act post-transcriptionally to repress co-transcriptional translation.

Question 5

How do transcription factors affect transcription?

Answer 5

• They act primarily to control transcription initiation.
• Activators can positively regulate expression. They promote the recruitment of RNA polymerase.
• Repressors can negatively regulate expression. They can block the recruitment of RNA polymerase.