W2 L2 The classic paradigm: Transcription Initiation by trans- acting Factors

Question 1

Gene expression and regulation

Answer 1

! Genes are expressed in order to generate products (RNA or protein) that have functional consequences for cells
! Gene expression can be measured at various levels
! Single gene, many genes, most genes or all genes (whole genome)
! Spatial or temporal
! Initiation or steady state
! Controlled changes in gene expression (ie. gene regulation) can also be measured at these various levels
! Difference between two or more conditions

Question 2

method of measuring gene regulation

Answer 2

-Transcription initiation
-RNA abundance
-RNA location
-Protein abundance
-Protein function
- Morphological phenotype

Question 3

Transcription genes state

Answer 3

! Constitutive: Expressed under all conditions usually for Core or essential genes
! Regulated: Expressed only under certain conditions for Genes required under special conditions
! Temporal and spatial
! Expressed at certain times or in certain cells/tissues.
! Negative control: Results in the DECREASE or turning OFF of gene transcription
! Positive control: Results in the INCREASE or turning ON of gene transcription

Question 4

E.coli and many carbon source

Answer 4

! Glucose is the preferred carbon source as it can easily be utilised for energy through glycolysis and the TCA cycle or assimilated into various compounds required for growth.
! Other carbon sources require conversion into glucose or other intermediates in order to be used for growth.

Question 5

Lac operon

Answer 5

! To utilise lactose as a carbon source, lactose is taken up from the environment and converted into glucose and galactose.
! Mutational analysis identified three closely linked genes which affect utilisation of lactose.
! lacY encodes a lactose permease (LacY)
! lacZ encodes a b-galactosidase (LacZ)
! lacI encodes a regulatory protein (LacI)
! The lacZ, lacY and lacA genes form an operon
! Transcribed as a polycistronic message
! Translated to produce the three enzymes
Everything is controlled by lacI

Question 6

Regulation of the lac Operon

Answer 6

! Expression of the lac operon (lacZ, lacY and lacA genes) is regulated by the environment.
! Transcription is high on lactose and low on glucose
! The system is INDUCIBLE.
! Lactose is converted to allolactose which serves as the actual inducer of the system.
! Lactose is catabolised by this system

Question 7

Genetic Dissection of lac Regulation

Answer 7

Question 8

Lac operon and RNA pol

Answer 8

! RNA polymerase is a multi-subunit complex that bind to the promoter region
! s component of RNA pol recognises the -35 and -10 sequences in the promoter

Question 9

Lac I and Lac operon

Answer 9

! Expression of the lac operon is negatively regulated by LacI.
! LacI is a DNA binding protein
! LacI binds the lacO in the absence of lactose
! Allolactose allosterically inhibits LacI binding

Question 10

CAP and lacOP

Answer 10

! Expression of the lac operon is positively regulated by CAP. Bind to promoter
! CAP is a DNA binding protein
! crp gene encodes the CAP (Catabolite Activator Protein)
! CAP interacts with cAMP (cyclic adenosine monophosphate)
! Glucose inhibits adenylate cyclase, blocking cAMP formation

Question 11

Types of DNA binding protein

Answer 11

! trans-acting regulatory genes (regulatory proteins or transcription factors)
! Positively acting (activation)
! Negatively acting (repression)
! cis-acting DNA binding sites
! Enhancers, upstream activation sites (UAS), upstream repression sites (URS), silencers

Question 12

how does DBP from far away work

Answer 12

-distal sequences attract each other via protein-protein interaction forming a pre-initiation complex

Question 13

Simple gene regulatory

Answer 13

-enhancer gene product bind to a promoter, leading to activation
-insulator can prevent enhancer to bind with promoter. Protein bound to insulator
-insulator can use to prevent one or multiple enhancer. Different enhancer can have different effect on the gene (temporal spatial)

Question 14

Enhancer acting in trans example

Answer 14

• enhancer can acts in trans (different chromosome)
  -odorant receptor have hundred of different receptor genes
  -one gene on in each different neuron (one receptor=one cell)
  -gene are on different chromosome but only one enhancer on Ch 14 (H enhancer)
• the H enhancer allow for only activation of one gene

Question 15

what does a transcriptional factor contain

Answer 15

• DNA binding domain
• Dimerisation domain for interaction and specificity
• Nuclear localisation domain
  -Interaction domain(s)

Question 16

DNA binding motif

Answer 16

-able to regconise the major groove of the DNA and the phosphate backbone

Question 17

different DNA binding motif

Answer 17

-Combinatorial control: Two DNA binding protein, three possible binding site

Question 18

Regulating the regulatory protein

Answer 18

• protein synthesis: changing the protein synthesis/ mRNA translation
• covalent modification: modification needed most common is phospolisation
• addition of second subunit needed
• unmasking: inhibitor bind to the regulator, need to be disassociate for the regulator to active
  -stimulation of nuclear entry: inhibitor prevent the transcription factor to be where it need to be
  -release from membrane: bind to membrane, cannot act until it is release

Question 19

Carbon and nitrogen: key components of biological processes

Answer 19

-Assimilation of preferred and alternate sources are tightly regulated, often at multiple levels
-carbon catabolism repression
-nitrogen metabolite repression

Question 20

Aspergillosis nitrogen metabolite repression

Answer 20

-AreA gene encodes a positively acting transcriptional regulator
-activates in the absence of NH4
-different ally nuclear localized (NLS and NES)
-controlled by import and export

Question 21

mRNA stability

Answer 21

-5’ cap and the poly A tail for stability via circularisation
-the loss of 5’ cap or gradual loss of A tail would eventually lead to degration

Question 22

mRNA stability of areA

Answer 22

• depend on he external environment, the mRNA post transcriptional modification would lead to it have a half life of 10 min or 40 min