L14: Regulation of transcription I

Question 1

Transcription factors (structure)

Answer 1

• Contain DNA binding domain, transactivating domain
• ~10% of genes in human genome code for TFs
• Types of DNA binding domain…
  Helix-turn helix, coiled coil, Zinc finger

Question 2

Targeted gene regulation in eukaroytes

Answer 2

• Most often at transcription initiation
• Also see it at elongation or termination
• Also see it in regulation from transcribed RNA itself
• Repressors or activators (less common) bind to operator

Question 3

Enhancer elements

Answer 3

• Found distal most frequently, either upstream or downstream from transcriptional start site
• Enable binding of transcription factors, that enable RNA pol to bind OR prevent RNA pol from binding

Question 4

Main division of genes in bacteria

Answer 4

• Constitutive/housekeeping: always expressed, e.g. for glucose metabolism
• Regulated: expressed only under certain circumstances e.g. changes in temperature or availability of nutrients
  -> typically only regulated at level of transcription

Question 5

Operon definition

Answer 5

• Expression of genes which encode proteins hat work together is coordinated by organisation of genes into operons
• In an operon, genes adjacent to each other are transcribed together into one polycistronic mRNA - which is then translated into separate proteins encoded by each gene in the operon
  e.g. Lac operon

Question 6

Lac operon (and regulation of its levels)

Answer 6

• Genes involved in utilising lactose
• When lactose is added as the sole carbon source, rapid increase in lac mRNA transcript (unstable)
• Rapid synthesis of enzymes required to metabolise lactose
  e.g. permease increases uptake of lactose into cell
  *allolactose; critical to regulation of lac operon

Question 7

Lac operon components + lac repressor

Answer 7

-Components of operon…
- lacZ: beta-galactidosase
- lacY:Permase
- lacA:Transacetylase
- The operon is transcribed to produce a single mRNA that is translated to produce three proteins
- lacI encodes lac repressor, situated immediately upstream. Has its own promoter an is constitutively expressed
-> NOT part of operon
-

Question 8

Lac repressor function (negative inducible regulation)

Answer 8

• When no lactose is present, LacI binds to lacO (the lac operator). No transcription occurs as RNA pol can’t bind)
• When lactose is present, allolactose binds to LacI, altering its shape. This allows transcription to occur (derepression)
• System is somewhat leaky (in rare circumstance, small amount of LacI not bound, so small amount of transcription, small amounts of allolactose
  -> gradual then rapid increase in lactose breakdown

Question 9

Lac operon levels in response to glucose (positive regulation)

Answer 9

• Presence of glucose inactivates adenylate cyclase and dramatically reduces levels of cAMP
• cAMP is an indicator of glucose levels (high when Gluc. low and vice versa)
• Catabolite activator protein (CAP) only binds to DNA in presence of cAMO, bends structure by 90^O
• CAP-cAMP activates gene expression; binding upstream of promoter facilitates RNA pol holoenzyme binding to its promoter
  -> maximal expression of lac operon when glucose levels low, otherwise you don’t get CAP binding, RNA pol binding v. inefficient

Question 10

Repressible regulation basic definition and example

Answer 10

• Operons for anabolic (biosynthetic) pathways turned off when end product is readily available (repressible regulation)
  e.g. trp operon

Question 11

Basic on/off regulation of trp operon

Answer 11

• 5 genes encoding enzymes for tryptophan biosynthesis are expressed in an operon as a polycistronic mRNA
  -> turned off by excess tryptophan
• Regulatory gene for trp operon is trpR, maps away from operon
• trpR encodes an aporepressor (protein which in initially synthesised state, can’t bind DNA)
  -> in absence of tryptophan, no binding of TrpR aporepressor to operator, transcription occurs
• In presence of excess tryptophan, aporepressor activated by binding tryptophan, binds to operator, prevents transcription
  …ON/OFF switch

Question 12

Expression of trp operon under tryptophan limitation

Answer 12

• Expression of trp but not maximal; controls length of full-length transcripts to short 140 bp transcripts terminated w/in trpL leader region
  -> attenuation
• Mechanism dependent on translation
• Secondary structures formed dependent on ribosome progression as mRNA translated
  -> attenuator structure (similar to type I terminator)
  -> termination of transcription prematurely (in presence of tryptophan)
• W/out attenuation, stalling of ribosome at Trp codons result in antitermination; transcription proceeds to produce full length mRNA encoding biosynthesis enzymes

Question 13

Riboswitches (about)

Answer 13

• RNA itself detects small molecule (e.g. adenine)
• Portions of transcript that directly bind
  -> controls RNA secondary structure regulating transcription or translation
• Two regions: aptamer (binds metabolite), expression platform (controls transcription or translation)

Question 14

Adenine riboswitch (B.subtilis)

Answer 14

• Regulates adenine synthesis and transport
• LOW adenine: regions 2 and 3 in RNA secondary structure form an anti-terminator, transcription proceeds
• HIGH adenine: regions 3 and 4 form a terminator