Principles of gene regulation

Question 1

Three experiments explain that different gene types share the same genome

Answer 1

1. nucleus from egg replaced by nucleus from skin cell. embryo still grows normally
2. chromosomes found in all cells
3. single cell from section of carrot forms an embryo and then full carrot

Question 2

Levels of gene regulation

Answer 2

transcription (most control at this level), RNA processing, RNA transport and localisation, translation control, mRNA degradation control, protein activity control

Question 3

Transcriptional units

Answer 3

simple gene (one protein per gene), operon (multiple proteins per polycistronic mRNA), operon with RNA processing, genes with polycistronic products (one mRNA leads to protein in number of forms)

Question 4

negative regulation

Answer 4

something happens to turn the gene off. e.g. lac operon and trp operon (removal of repressor protein switches gene on)

Question 5

positive regulation

Answer 5

something happens to turn the gene on (e.g. cap and the lac operon)

Question 6

GRP basics

Answer 6

positive and negative regulation, combine to alter outcome e.g. individual binding, dna directed complexes. Master GRPs can effect multiple genes in different ways. Bind at enhancer and UPEs

Question 7

Prokaryote consensus sequence

Answer 7

promotor sequence - strong promotors have more matching sequence. (-35TTGACA -10TATAAT)

Question 8

CAP and the lac operon

Answer 8

Low glucose = hich cAMP, binds to CAP, CAP binds promotor, loads polymerase (glucose and cap is positive regulation)

Question 9

Nitrogen metabolism

Answer 9

deficiency, alpha-ketoglutarate to glutamine ratio rises, activates uridylating enzyme, forms PII pump, dissociates PII subunit from ntrB, activates ntrB kinase, forms ntrC phosphate, activates gene transcription.

Question 10

ntrC

Answer 10

Upstream enhancer. Is a GAP. When phophorylated activates the gene via a looped activation intermediate

Question 11

Sigma factors altering

Answer 11

Under certain environmental conditions sigma factors are used that recognise different promoter sequences. e.g. heat shock and sigma54 (ntrA gene) in nitrogen limitation

Question 12

Eukaryotic transcriptional control at initiation

Answer 12

control of transcription by transcription factors

Question 13

control regions in eukaryote

Answer 13

promotor, (TATA box -25), upstream promotor elements (sequence of DNA that must be in a relatively fixed position relative to the start of transcription. It can include the TATA box but excludes an enhancer) and enhancer (distant not fixed)

Question 14

enhancer characteristics

Answer 14

they exert strong activation of transcription of a linked gene from the correct start site. ● They activate transcription when placed in either orientation with respect to linked genes. ● They are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site. ● They exert preferential stimulation of the closest of two tandem promoters. First observed in SV40

Question 15

three experiments to identify GRP binding domains

Answer 15

characterise the enhancer region. Mutational analysis, footprinting and gel retardation

Question 16

Mutational analysis

Answer 16

Take beta-globin enhancer , 108 nucleotides long. Cause mutation every 4th base, 27 different enhancers. Put into test plasmid. Reporter gene (CAT) is produced and gives indication of beta globin promotion. Placed in leaky erythrocyte. Those with low activation show which bp are important

Question 17

Footprinting

Answer 17

Labelled enhancer at one end. Limited digestion performed. Cut at every base pair, gives ladder of fragments. second solution: Enhancer mixed with nuclear extract of GRP. Perform digest. Gap in ladder is where the DNAse cant cut because GRP is bound.

Question 18

Gel retardation

Answer 18

Labelled enhancer fragment. Cut it up and add protein. When enhancer is bound the extract moves less far (retarded). This sample or band should be extracted and sent for analysis/ protein identification.

Question 19

Enhancer/UPE control

Answer 19

Modular design, synergistic activity, small number of GRPs. Enhancer binds to promoter and increases or decreases transcription

Question 20

GRPs mechanism

Answer 20

DNA binding domain and activation domain. In domain swap experiments the GRP is inactive with wrong DNA binding domain because it cannot bind its recognition sequence. (e.g. gal4 and lexa)

Question 21

Intracellular GRP example

Answer 21

hormone (e.g. steroid) binds to GRP and displaces inhibitory protein, DNA binding site is therfore made available. Binds to enhancer which activates the promoter and leads to transcription

Question 22

Binding domains

Answer 22

helix-turn-helix, C2H2 zinc finger, leucine zipper, helix loop helix

Question 23

Helix turn helix

Answer 23

Typical of homeotic domains – turning one body part into another. generally alpha helices seperated by a beta turn

Question 24

C2H2 zinc finger

Answer 24

a loop of 12 amino acids anchored by two cysteine and two histidine residues that tetrahedrally co-ordinate a zinc ion. This motif folds into a compact structure comprising two -strands and one -helix, the latter binding in the major groove of DNA. The alpha-helical region contains conserved basic amino acids which are responsible for interacting with the DNA. Usually, three or more C2H2 zinc fingers are required for DNA binding

Question 25

leucine zipper

Answer 25

hydrophobic faces of alpha helical regions interact. leucine every seven amino acids form a leucine zipper. Dimer is formed. Two alpha helices (one from each dimer) form the basic region which binds to the DNA like a clamp.

Question 26

helix loop helix

Answer 26

The overall structure of this domain is similar to the leucine zipper, except that a nonhelical loop of polypeptide chain separates two -helices in each mono- meric protein. Hydrophobic residues on one side of the C-terminal -helix allow dimerization. This structure is found in the MyoD (belongs to a family of proteins known as myogenic regulatory factors) and master gene regulators. Needs to dimerise with basic dimer to bind DNA.

Question 27

Molecular Mechanisms that Generate Different Cell Types

Answer 27

DNA rearrangements (phase variation, the MAT locus) GRPs acting as switches (lamda bacteriophage and similar switches in eukaryotes), chromatin structure (position effect variegation, chromosomal position effects, locus control region), Methylation (gene expression, CG islands)

Question 28

Phase variation

Answer 28

DNA rearrangement. Salmonella change flagellins (h1 and h2) to deal with immune response. H2 is produced via polycistronic Mrna but when promoter is inverted h1 is produced.
This switch between phenotypic traits = phase variation
Inversion is caused by HIN which works on a HIX site. FIS controls the sytem, acting as an enhancer. It binds to enhancer, brings HIX into complex that is recognised by HIN, which cuts at the HIX sites:
HIX (12bp—2bp—12bp)————-inverted sequence——–(12bp—2bp—12bp)hix

Question 29

MAT

Answer 29

mat - mating locus of yeast. an example of Combinatorial Gene Control. refer to slide. a produces mata1 no effect (asg turned on by mcm1 hsg always on, alphasg off), alpha produces matalpha1 and 2 inhibits mcm1 (asg turned off by inhibition of mat alpha2 alphasg turned on by mcm1 and matalpha1 hsg on). alpha and a together diploid cell (mata1 and matalpha2 combine to turn hsg off, asg turned off by mcm1 and matalpha2 and alphasg turned off.

Question 30

Cassette switching

Answer 30

Once HO cuts the DNA at MAT, exonucleases are attracted to the cut DNA ends and begin to degrade the DNA on both sides of the cut site. This DNA degradation by exonucleases eliminates the DNA which encoded the MAT allele; however, the resulting gap in the DNA is repaired by copying in the genetic information present at either HML or HMR, filling in a new allele of either the MATa or MATα gene. Thus, the silenced alleles of MATa and MATα present at HML and HMR serve as a source of genetic information to repair the HO-induced DNA damage at the active MAT locus. Ability to switch mating type is asymmetrically inherited because only mother cells have the ability to produce Ho (because they have been through G1). EL and ER flank the three loci to make them silent. They are negative enhancers. SIR (silent information regulatory protein) is a grp that inhibits HO and closes down dna by binding to EL and ER regions

Question 31

Lamda bacteriophage

Answer 31

CI (lytic stage) and Cro (prophage stage) act as switches between integration and lysis, controlled by environmental conditions. GRPs act directly as switches in this example

Question 32

Position-effect variegation

Answer 32

a variegation caused by the inactivation of a gene in some cells through its abnormal juxtaposition with heterochromatin. heterochromatin may spread to euchromatin and make it inactive. inversion in drosophilia produces white eye mutation because the white gene is nearer to the heterochromatin

Question 33

chromosome position effect

Answer 33

used to describe the variation of expression exhibited by identical transgenes that insert into different regions of a genome. possibly due to different enhancers

Question 34

LCR

Answer 34

Locus control regions (LCR) are defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number-dependent manner at abnmormal chromatin sites. If LCR is deleted, chromatin is condensed and globin genes switch off

Question 35

LCR loop model

Answer 35

gene is turned on after chromatin is decondensed. a section of the DNA loops around to different extent over the desired gene of expression. Serves as entry site for protein which catalyses the alteration of chromatin, serves as site for gyration of constrained chromatin and superhelical tension alters structure, serves as nucleating site for assembly/dissasembly of chromatin protein coat leading to change in structure

Question 36

Methylation

Answer 36

Mehtylation is restricted to CG when they fall next to each other in the sequence.
Pattern is maintained by maintenance methylase and inherited. In an experiment unmethylated DNA was added to fertilised egg and all CGs became methylated with 1 exception. Therefore DNA starts off very methylated. likely to control GRPs. In experiment methylated and unmethylated DNA added to muscle cells and fibroblast. In muscle cells both genes were still converted at same rate however in fibroblast their was no expression for methylation and some for unmethylated. This shows that methylation reinforces the action of GRPs. Makes genes less leaky

Question 37

CG islands

Answer 37

CGs are methylated. When they are deaminated the C becomes a T and it is not repaired. Many CG pairs are therefore lost. However, the areas around promoters of active housekeeping genes are protected by GRPs and so CG islands are formed and maintained.

Question 38

Post transcriptional controls

Answer 38

attenuation, alternative splicing, rna transport control. tanslational control, translational frameshifting and RNA stability

Question 39

Attenuation (e.g. in trp operon)

Answer 39

The leader sequence of the trp operon RNA contains four regions of complementary sequence which can form different base-paired RNA structures. These are termed sequences 1, 2, 3 and 4. The attenuator hairpin is the product of the base pairing of sequences 3 and 4 (3:4 structure). Sequences 1 and 2 are also complementary and can form a second 1:2 hairpin. However, sequence 2 is also complementary to sequence 3. If sequences 2 and 3 form a 2:3 hairpin structure, the 3:4 attenuator hairpin cannot be formed and transcription termination will not occur. Under normal conditions, the formation of the 1:2 and 3:4 hairpins is energetically favorable. under low trp the 2:3 will form and polymerase will continue transcription

Question 40

alternate splicing

Answer 40

different splicing of exons and exons Constitutive- random collection of sites – usually not a working protein
Regulative – controlled by self (e.g negatively by repressors or positively by activators). Happens in Src gene of chickens to produce gene found in most tissues or in neural tissues

Question 41

Fly sex selection

Answer 41

males x chrom/autosome ratio = 0.5. Sxl gene produces non-function gene, tra gene produces non-functional gene, inactive splice site on dsx produces protein that represses female genes. Females x chrom/autosome ratio = 1. Sxl gene produces protein that blocks splice site on sxl and tra, tra produces protein that (with tra-2) activates splice site on dsx produces protein with 30aa that are female specific and inhibit male genes.

Question 42

antibody secretion

Answer 42

alternate splicing of short and long transcripts produces membrane bound or secreted antibodies

Question 43

RNA control

Answer 43

RNA export is an active process that may require Specific RNA recognition signals in nuclear membrane, All RNAs transported unless specifically retained, Combination of 1 and 2 ie selective export and selective retention. Small amounts of RNA leave the nucleus, may vary between cell types

Question 44

Translational control

Answer 44

repressor protein binds to 5’ sequence and turns off transcription e.g. Iconrotase binds IRE loop in iron starvation and prevents ferotin from storing iron. < NEGATIVE. POSITIVE> Enhancers act on ribosomal binding sites. often in viruses a viral sequences is entered into the DNA and more viral protein is produced

Question 45

translational frameshifting

Answer 45

Normally a protein is translated from a template mRNA with consecutive blocks of 3 nucleotides being read as single amino acids. However, certain organisms may exhibit a change or shift in the ribosomes frame by one or two nucleotides when translating the genetic code. This happens commonly in viruses. tRNA can slip a base back because of base wobble. mRNA structure may effect it. E.g. GAG formed or -1 frameshift following leucine incorporation leads to GAG-POL fusion protein

Question 46

RNA stability

Answer 46

RNAs made more stable by polyA. If histone 3’ tail added to rna then the synthesis becomes modulated by cell cycle instability. An evolutionary conservered 50 nucleotide AU rich region in untranslated region of 3’ mRNA causes instability. Cytosolic aconitase in iron starvation. Stabilises transferrin mRNA and transferrin is made but destabalises when iron is high (fe binds it and dissociates it from mRNA)