Genetic Regulation

Question 1

Why regulate genes?

Answer 1

• gene expression is costly and natural selection has shaped gene architecture to reduce this cost
• the more a gene is expressed the more selection pressure it is under to be optimised
  cost-efficiency: less mRNA and high initiation, slow translation speed, hydrophilic aa, cheap aa

Question 2

Examples of regulation

Answer 2

• differential gene expression patterns define cell development into different cell types (eg. intestinal cycle)
• different environments require difference gene sets-cost efficient adaptation to the environment (eg. biofilms switching between sessile and motile lifestyles)

Question 3

Principles of Regulation

Answer 3

1. DNA rearrangment
2. transcriptional regulation
   - splicing, mRNA stability
3. post-transcriptional regulation
   - miRNA, mRNA stability, riboswitches

Question 4

Elements of regulation

Answer 4

cis: DNA sequences controlling gene expression
- promotor regulatory sequence
trans: proteins binding control regions to stimulate/inhibit gene expression
- RNAP regulator

Question 5

cis-elements

Answer 5

• binding site for repressors or promotor elements
• regions of DNA to which these regulators bind located on the same molecule of DNA as the gene whose expression it regulates
• usually tandem repeats

Question 6

trans-elements

Answer 6

• binding elements like RNA polymerase forming transcription bubble
• transcription factors binding control regions to stimulate or inhibit gene expression
• diffusible factors acting on a different molecule of DNA from where they are encoded
• sensing - oligomerisatoin - DNA binding

Question 7

Positive Regulation

Answer 7

• signal molecule changes activator conformation to allow binding to DNA and promotion of transcription

Question 8

Negative Regulation

Answer 8

1. repressor already bound and signal causes dissociation

2. repressor binds when triggered into a conformational change by a signal

Question 9

DNA footprinting

Answer 9

1. PCR amplify and label the region of interest, which is a predicted protein-binding site.
2. Add the protein under investigation to the labelled amplified DNA. Make a control tube with just DNA and no protein.
3. Immunoprecipitate the protein bound DNA.
4. Add a modifying or cleavage agent.
5. Run both samples by polyacrylamide gel electrophoresis (PAGE).
6. The control sample will form a uniform ladder, whereas the test will show gaps in the pattern where the protein protects the DNA (if it binds).
7. Do a comparative analysis to determine the exact sequence where the protein is bound.

‘footprint’ is where no cleavage is observed and electrophoresis shows dark bands that indicate the presence of larger uncut fragments compared to normal cut bands - these are regions are bound by RNAP

Question 10

Reporter Gene Assay: promoter mapping

Answer 10

The main purpose of the reporter gene assay is to investigate the promoter of a gene of interest, i.e. the regulation of its expression. This can be done by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyses a reaction that produces light.

Usually, the cells are then exposed to different factors or conditions, or changes can be made in the order of the reporter, the effect of which can be easily tracked by measuring changes in light emission.

• amplify region of interest and ligate with reporter gene
• deletion of region between nucleotides reduces expression (measured by luminescence) but doesn’t inhibit expression - likely cis element allowing activator binding

Question 11

DNA affinity chromatography

Answer 11

1. cell proteins passed through column containing DNA of many different sequences
2. low salt wash removes proteins not binding DNA
3. medium salt wash elutes many different DNA binding proteins
4. DNA binding proteins from process 1 passed through column with sequence specific matrix
5. medium salt wash removes all proteins not specific no this sequence
6. high salt wash elutes rare protein that specifically recognises this sequence

Question 12

Gel Shift Assay

Answer 12

The control lane (DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, assuming that the protein is capable of binding to the fragment, the lane with a protein that binds present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to protein which is ‘shifted’ up on the gel (since it has moved more slowly).
Binding of the trans element to the DNA causes a shift in mobility - the protein DNA complex migrates slower through a PAGE gel than bare DNA

Question 13

Operon Architecture

Answer 13

• linear sequences of DNA containing a promotor, a terminator, an operator (repressor protein binding site), repressor, and activator
• operons are controlled by regulatory genes found elsewhere on the chromosome, which regulate the expression of the structural genes in response to an environmental signal

Question 14

Evolution of Operons

Answer 14

• horizontal gene transfer

- by combining transcription and translation synthesis and assembly are localised

Question 15

lac operon

Answer 15

• sigma 70 regulated operon
• negative regulation and catabolite repression
• no lactose causes the repressor protein lacI to bind to the operator site and blocks RNAP
• lacI is a dimer protein looping the promoter region to block access
• lactose binds to lacI, preventing its attachment to the operator and RNAP transcribes DNA

Question 16

Catabolite Repression

Answer 16

• glucose inhibits activity of adenylyl cyclase which catalyses production of cAMP from ATP
• energetically favorable to use glucose up first
• low cAMP means CAP protein doesn’t bind to the DNA so operon is not transcribed
• lactose allows activation of the enzyme so the the cAMP-CAP complex binds to DNA and operon is transcribed

Question 17

How does glucose inhibit AC??

Answer 17

1: High glucose - PTS transfers phosphate to glucose (system saturated) - PTS low phosphorylation - no interaction with adenylyl cyclase - Low cAMP
2: High glucose - High ATP (more efficient energy production) - low adenylyl cyclase and low cAMP - more anabolism

Question 18

Single molecule trigger

Answer 18

• lac operon induction by lactose in single cells is governed by a single molecule trigger
• cells with 1 lacZ expressed at the moment of the switch will respond readily
• cells without the lac proteins are sensorless and must wait for transcriptional noise (random burst of lac expression)
• population response to galactose delayed

Question 19

Arabinose Operon

Answer 19

• control gene (araC), control sites (O,I) and structural genes (araB,A,D)
• B,A,D are required for arabinose breakdown
• C is an activator and repressor
  cis: araO, araI and CAP binding site
  trans: araC and cAMP-CAP

Question 20

Types of Arabinose Operon Regulation

Answer 20

negative regulation: araC binds to O and I
positive regulation: araC binds to I
catabolite repression: glucose reduces cAMP levels
negative autoregulation: high levels of araC bind to O to inhibit its own expression

Question 21

Arabinose Operon Expression

Answer 21

no araC: RNAP free to transcribe araC
high glucose, low arabinose: negative regulation by araC: elongated C binds at I and O to loop DNA and RNAP cannot interact to make araBAD
low glucose, high arabinose: postitive regulation by araC: compact araC binds at I and allows araBAD transcription

Question 22

AraC autoregulation

Answer 22

• high araC levels
• binds to araO
• RNAP can’t approach Parac promoter and transcription of araC is suppressed

Question 23

Tryptophan Operon

Answer 23

negative regulation with Trp repressor and attenuation

5 genes encoded next to each other

Question 24

Attenuation

Answer 24

Reduces expression of the trp operon when levels of tryptophan are high by preventing completion of transcription. When levels of tryptophan are high, attenuation causes RNA polymerase to stop prematurely when it’s transcribing the trp operon.

• low trp = ribosome stalls at 2 trp codons and stem loop between 2/3 can form not affecting transcription
• high trp = stem loop between 3/4 can form terminating transcription (RNAP stalls at attenuator and dissociates)

Question 25

Repression of Tryptophan operon

Answer 25

When bound to tryptophan, the trp repressor blocks expression of the operon.. - The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active (corepressor). - When there is little tryptophan in the cell, on the other hand, the trp repressor is inactive (because no tryptophan is available to bind to and activate it). It does not attach to the DNA or block transcription, and this allows the trp operon to be transcribed by RNA polymerase.

Question 26

Riboswitches

Answer 26

- Riboswitches are gene control elements that directly bind to specific ligands to regulate gene expression without the need for proteins-they act in cis (cis elements).  - They are examples of negative regulation, where the presence of the molecule leads to termination of expression. - Only one eukaryotic riboswitch that senses thiamine pyrophosphate (TPP) that works via alternative splicing (the mechanism of inhibiting translation initiation would not work in eukaryotes, similar to attenuation). - translation initiation: small molecule binding to a riboswitch triggers formation of a hairpin loop containing the site where ribosomes normally bind

Question 27

Epigenetics

Answer 27

- heritable alterations in gene expression that do not arise from altered DNA sequence, usually changes in chromatin like methylation, histone modification, and nucleosome positioning

Question 28

Histone Modification

Answer 28

Modification of histone tails - acetyltransferase adds acetyl groups to open chromatin and activate transcription - deacetylase removes acetyl groups and condenses chromatin to repress transcription Histones are highly basic and positively charged proteins binding DNA phosphates and prevent transcription. When acetylated, histones lose their charge and dissociate DNA

Question 29

DNA methylation

Answer 29

- methylation of CpG island represses gene expression - prevents transcription factor binding - methyl-CpG binding protein recruits histone deacetylase and condenses chromatin

Question 30

Eukaryotic regulatory elements

Answer 30

1. promoter 2. promoter proximal elements 3. enhancers

Question 31

RNAP II

Answer 31

- forms pre-initiation complex | - mediator modulates TFIIH activity

Question 32

Gal Regulation System

Answer 32

- Gal genes only need expression when galactose is present - Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription. - Gal80 blocks Gal4 from activating transcription - also has catabolite repression

Question 33

Gal Regulation in Yeast

Answer 33

1. galactose absence, Gal80 blocks gal4 from activating transcription 2. galactose is present and binds to gal3 and brings about a change in the conformation of gal80 3. gal4 interacts with basal transcription apparatus and stimulates transcription

Question 34

Gal4-UAS system in Drosophila

Answer 34

1. gal4 lines express gal4 in subset of animal's tissues 2. reporter lines of fruit flies express UAS region next to gene of interest 3. in certain offspring the desired tissue will express the gene of interest - choose promoter based on tissue of interest - Since Gal4 by itself is not visible, and has little effect on cells, the other necessary part of this system are the "reporter lines". These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells that are producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein

Question 35

pre-mRNA structure

Answer 35

- coding sequences (exons) interrupted by intervening sequences (introns) - one gene can code for multiple isoforms of a protein

Question 36

Splicing

Answer 36

- snRNPs are RNA protein compexes 1. lariat formation and 5' splice site cleavage 2. 3' splice site cleavage and exon sequence ligation

Question 37

micro-RNA

Answer 37

- miRNAs are transcribed by RNAP II - small single-stranded non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. - bind to the 3'UTR sequence on mRNA of target untranslated mRNA to repress protein production and destabilize mRNA

Question 38

Lin-4 micro-RNA regulation

Answer 38

- lin-4: miRNA required for C. elegans development - lin-4-/- mutants and mutants where the lin-4 binding site in the lin-14 UTR has been mutated fail to turn off lin-14 expression at the L2 stage and both mutants have retarded development. - lin-4 is required for L1→ L2 transition. - lin-14 is a target gene of lin-4 and the drop in lin-14 protein caused by lin-4 is required for L1→ L2 transition. - lin-14 mRNA levels remain constant throughout development Lin-4 is a miRNA acting by antisense base pairing to 7 complementary elements in the lin-14 mRNA 3' UTR to down-regulate lin-14 - Binding of lin-4 to lin-14 blocks protein synthesis after the initiation of translation.