Bacteriology - Part III (Gene regulation)

Question 1

RNA polymerase in Bacteria

Answer 1

-RNA Polymerase (RNAP) transcribes the mRNA from DNA in bacteria -4 catalytic subunits -Regulatory sigma subunit -Only 1 RNApol in bacteria -Transcription initiates when the RNAP complex bins the promoter/operon

Question 2

Transcription and Translation relationship in bacteria

Answer 2

Transcription and translation are coupled – ribosome can make proteins from mRNA that are still being translated

Question 3

Bacterial transcriptional activators

Answer 3

• Bind to sequences at or near the promotor region
• Help to recruit RNApol to the operator by binding the promoter region, usually upstream of the operator

Question 4

Bacterial transcriptional Repressors

Answer 4

Bind in the operator region/consensus sequence and prevents the binding of RNApol to the operator and prevents transcription

Question 5

Lac operon

Answer 5

-Polycistronic set of genes that encode for proteins that metabolize lactose

Question 6

LacI is?

Answer 6

-LacI encode the lac repressor which prevent transcription of the operon (Constitutively active in its expression)

Question 7

LacI inhibition

Answer 7

When Lac repressor binds to lactose it is then inactivated and no longer able to bind the promoter, thus allows the transcription of laxZYA to proceed >>> presence of lactose induce lac operon expression

Question 8

Attenuation is?

Answer 8

Premature termination of transcription or translation based on different secondary structures of mRNA

Question 9

Example of Attenuation

Answer 9

Trp operon

Has two levels of control

• Standard repressor (bound to trp = inactive)
• Attenuation control

Question 10

Trp Attenuation

Answer 10

• Attenuator region has high level of Trp codon – located downstream of the operator but upstream of the trp genes themselves
• High trp levels form stem-loop pairings (1:2 and 3:4) (mRNA secondary structure) cause the early termination of transcription BEFORE Trp genes are reached – thus preventing the synthesis of proteins needed for Trp synthesis
• Essentially the ribosome reaches the trp codons and quickly adds the trp AA to the protein. Because this occurs quickly the ribosome blocks the pairing of 2 and 3. Thus 3&4 pair and effectively stop transcription.

Question 11

Trp Inhibition of Attenuation (i.e. proceeding as normal)

Answer 11

• Low trp level allows the stem-loop (2:3) that allows for transcription to proceeded as normal – Ribosome is stalled via the 2:3 stem loop which prevents the 3:4 stem loop from forming and causing early transcription termination >>> transcription can continue and transcribe the rest of the operon
• Ribosome moves more slowly at the trp codons due to low concentration, this blocks 1 from pairing with 2, thus 2 pairs with 3 and transcription does not get terminated.

Question 12

Riboswitch

Answer 12

• RNA genetic switch that binds metabolites without the needed involvement of proteins
• Located in the 5’ UTR of the mRNA
• Usually synthesized first so it can respond and regulate the mRNA before the entire thing is produced.

Question 13

Riboswitch mechanism

Answer 13

• Binding of the ligand to the switch causes a conformational change that changes an Anti-Terminator stem-loop to a Terminator – prevent transcription
• Binding to the riboswitch sequesters the Shin Delgarno sequence (stem loop needed for transcription) and forms an Anti-Shine Delgarno – thus preventing transcription

Question 14

Example of ligand binding to riboswitch preventing transcription

Answer 14

-Binding of thiamine pyrophosphate to a riboswitch prevents the transcription of the genes needed to synthesize thiamine >>> i.e. high thiamine levels inhibit its own transcription.

Question 15

Iron regulation in E. Coli

Answer 15

• Excess Iron prevents transcription of Fe acquisiton genes by binding the Fur repressor
• Low Iron causes Fur to unbind and therefore Fe acquisition genes are trasncribed