Regulation of Transcription

Question 1

What are cis acting and transacting sequences?

Answer 1

Transcription of a gene is regulated by transacting and cis acting sequences

Cis acting Sequences: affect expression due to being physically connected to the sequence they regulate

• promoter: binds to transcription factors to bring RNAP close to a structural sequence, so makes it more likely that structural sequence will be transcribed.
• Operator: is where regulatory proteins bind
• Enhancers: increase the likelihood that transcription of a particular gene will occur
• Silencers: bind to repressors - prevent transcription of a gene (e.g inhibitors)

Trans acting Sequences: regulate other sequences without necessarily being physically close to the regulated sequence

• regulatory proteins are transacting and must be synthesised themselves (from their own gene with promoter and potentially operator regions) - can regulate the regulator

Question 2

What is positive and negative regulation of transcription?

Answer 2

Positive Regulation - protein/DNA interaction increases transcription
- activator binds to operator. promoter able to bind to RNAP

Negative Regulation - protein/DNA interaction decreases transcription

• co repressor binds to promoter, preventing RNAP from binding
• Repressor binds to operator

Question 3

What is repressible and inducible control of transcription?

Answer 3

Repressible = transcription is ON by default
- turned OFF by a REPRESSOR

Inducible = transcription is OFF by default
- turned ON by INDUCER (small molecule whose presence helps increase transcription through allosteric effect on the protein)

Question 4

What are enhancers?

Answer 4

Enhancers are cis-acting sequence which recruit transcription factors which increase the likelihood of transcription occurring by positively interacting with RNAP and Basal TF
- can be located upstream/downstream - thousands of nt away or even inside the gene.

• DNA bending protein -> allow enhancer regions to interact with promoter

Question 5

What are operons?

Answer 5

Operons are clusters of genes that share the same promoter and are transcribed as a single large mRNA that contains multiple structural genes or cistrons.

Question 6

What is the trp operon in E.Coli?

Answer 6

TRP operon found in E.Coli bacteria
- group of polycistronic genes that encode for biosynthetic enzymes for amino acid tryptophan

• under negative repressible control
  negative = transcription is turned OFF when protein binds to DNA
  repressible = transcription is ON by default unless corepressor (trp) is added

trp repressor (Repressor regulatory protein, TrpR) acts as both a sensor and a switch.

When tryptophan levels are HIGH:
- tryptophan binds to tryptophan repressor -> becomes active and binds to operator so transcription of TRP operon is OFF

When tryptophan levels are LOW:
- no tryptophan bind to repressor -> repressor inactive -> allows transcription to be oN

Question 7

How does attenuation regulate Trp operon?

Answer 7

Attenuation of TRP Operon: rather than blocking initiation of transcription like trp repressor, attenuation prevents completion of transcription

• occurs in prokaryotes as transcription and translation are concurrent (occur in the same place)

TRP operon has a leader sequence called Trp L which codes for 4 sequences:

• sequence 1 = codes for a leader peptide which requires 2 tryptophans (UUGUUG)
• sequence 2 = contains a string of bases that are complementary to string of bases in sequence 3
• Sequence 3 = contains a string of bases that are complementary to bases in sequence 4

when tryptophan levels are HIGH - high trp tRNA

• ribosome reads through 1 easily and shields 2
• therefore 3 and 4 form transcription terminator hairpin

When tryptophan levels are LOW - low trp tRNA

• Ribosome stalls in 1 waiting for trp tRNA
• therefore 2 binds to 3 -> anti terminator hair pin
• so 3 and 4 cannot form transcription terminator hairpin

Question 8

What are lac operons in E.Coli?

Answer 8

Lac operons: contains genes involved in lactose metabolism. It’s expressed only when lactose is present and glucose is absent.
- lac operon contains 3 genes: LacZ, LacY, LacA

• under positive control by CAP/cAMP
• under negative inducible control
  Inducible = transcription is OFF unless we add inducer (Allolactose)
  Negative = transcription is turned OFF when protein binds to DNA

When repressor regulatory protein (LacI) binds to inducer (allolactose), repressor binds to operator and blocks transcription

NO LACTOSE:
LacI (Lac repressor) binds to operator and gets in the way of RNAP, preventing transcription

WITH LACTOSE:
Allolactose (Isomer of lactose) binds to LacI and makes it release operator so RNAP can continue with transcription

Question 9

How does catabolite activator proteins regulate Lac operon?

Answer 9

Lac operon under POSITIVE control by Catabolite Activator Proteins (CAP)

CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription.

LOW [GLUCOSE] → increased cAMP production
→ cAMP binds to CAP → CAP binds to CAP binding site (CBS) → increases recruitment of RNAP → increases transcription of lac enzymes

HIGH [GLUCOSE] → no cAMP production
→ no cAMP binds to CAP → CAP cannot bind to DNA → transcription occurs at low level

Question 10

How are regulator proteins regulated?

Answer 10

Regulator proteins are themselves regulated

- LacI can repress its own transcription