Genetics Midterm #2

Question 1

Transcriptional Regulation

Answer 1

a. Regulatory proteins and transcription factors bind to consensus DNA sequences (promoter regions) to facilitate transcription.
b. Additional regulatory DNA sequences (enhancers and silencers) bind regulatory proteins to facilitate transcription of specific genes in each cell type.
c. Open chromatin structure is favorable for transcription formed by protein action.
d. Alternative promoters are utilized in different cell types to produce different pre-mRNA molecules.
e. Methylation of DNA inhibits transcription.

Question 2

mRNA Processing

Answer 2

a. Capping of the 5’ end, polyadenylation of the 3’ end, and intron splicing modify pre-mRNA.
b. Alternative capping and polyadenylation sites can be used in different cell types.
c. Alternative splicing produces different mature mRNA molecules from some cell types.
d. RNA editing modifies the base sequences of mRNA.

Question 3

Regulation of mature mRNA

Answer 3

a. Translational regulatory proteins bind mature mRNA to delay translation initiation.
b. Small RNAs regulate the stability or translation initiation.
c. Transport of mature mRNA to cytoplasm is regulated.
d. RNA stability is regulated.

Question 4

Cis-Acting Regulatory Sequences Bind Trans-Acting Regulatory Proteins to Control Eukaryotic Transcription.

Answer 4

• Activator proteins bind regulatory sequences to stimulate transcription.
• Repressor proteins bind other sequences to hinder transcription.
• Regulatory proteins are often found in large complexes in eukaryotes, unlike in bacteria.
• Individual transcription factors may regulate tens to hundreds of target genes.

Question 5

Integration and Modularity of Eukaryotic Regulatory Sequences

Answer 5

• Enhancers and silencers typically contain binding sites (modules) for a number of transcription factors
• The modules allow the enhancers and silencers to integrate activities of different transcription factors to produce different outputs.
• Pioneer factors are the first to bind regulatory sequences, facilitating binding of additional transcription factors

Question 6

Modularity of Regulatory Sequences:

Answer 6

• Multiple proteins bind, interact, and recruit additional proteins.
• Note: Identity of proteins bound can convert an enhancer into a silencer…
• Two proteins might bind cooperatively or, conversely, might sterically hinder one another’s binding.

Question 7

Transcription Factor Synthesis

Answer 7

• Transcription factor availability is dependent on their transcription and translation in the cell.
• Synthesis of transcription factors is tightly regulated and different cell types have distinct arrays of transcription factors.
• The availability of some transcription factors is controlled through signal transduction.

Question 8

Signal Transduction

Answer 8

• Signal transduction pathways: events outside the cell stimulate sequential events inside the cell that result in new gene expression patterns.
• Involve transmembrane proteins that receive signals externally through an extracellular interaction domain.
• They transmit signals within the cell via a binding domain inside the cell.

Question 9

Process of External Signaling into an Internal Response and the Effect on gene transcription

Answer 9

1. Stimulus
2. Release of transcription factor protein
3. Activation of transcription factor
4. Transportation to nucleus
5. Enhancer binding
6. Transcription initiation.

Question 10

Identifying Eukaryotic Gene Regulatory Sequences: How do we identify control elements like enhancers?

Answer 10

Just like we did to identify promoter elements!
Example:
a. clone gene of interest into plasmid.
b. test the cloned gene’s activity in appropriate (and inappropriate) cell types.
c. Delete blocks of DNA upstream and downstream coding sequences to ask if they are essential to appropriate expression.
d. Deletion– loss of high expression? Now test if that region can act as an enhancer..

Question 11

Have identified enhancer (by activity)

Answer 11

Now identify proteins that make it work:

• Gel shift
• Footprinting
• DNA sequence (look for conserved motifs already described in other enhancers/promoters)
• ChIP (chromatin immunoprecipitation)

Question 12

Band Shift Assay

Answer 12

Identifies DNA fragments that bind nuclear proteins (usually extract from cell)

• Add antibody to the suspected Transcription Factor.
• “Super shift” results (if Ab binds TF bound to DNA)

Question 13

DNA footprint protection Assay

Answer 13

Assay shows you WHERE on the fragment the protein is binding.

Question 14

ChIP -Chromatin Immunoprecipitation

Answer 14

• You know a protein can bind a promoter/enhancer element in vitro.
• Does the protein bind that element in vivo? (in living cells)
• Isolate nuclei; isolate chromatin; Cross-link DNA-bound proteins to DNA; shear DNA; use antibody to precipitate protein; reverse cross-link to elute bound DNA; PCR to see if eluted DNA contains element.
• If gene is expressed only in fibroblasts, do you expect ChIP results will be the same in fibroblasts and lymphocytes?

Question 15

Locus Control Regions

Answer 15

A locus control region (LCR) is a highly specialized regulatory sequence that acts to not only enhance transcription but also to insulate a locus from the effects of neighboring genes.
- First LCR was discovered in human beta-globin locus.

Question 16

Hemoglobin has…

Answer 16

2 alpha-globin polypeptides
2 beta-globin polypeptides

• There are different “forms” of beta-like globin polypeptide made and incorporated into hemoglobin during development.

Question 17

Beta-globin-gene complex

Answer 17

• Has “different” forms of beta-like globin polypeptides made during development.
• Each form has one gene
• Each gene has its own promoter…

Question 18

Locus Control Region - How was it discovered?

Answer 18

• Thalassemias: loss/mutation of alpha-globin coding sequences. loss/mutation of beta-globin coding sequences. No mutation of either alpha or beta-globin coding sequences– but still no hemoglobin???
• “Transgenic animals”: introduce a gene into the genome of an embryo– is incorporated into cells that become germ cells– “transgene” is passed to next generation (in ALL progeny cells)
• “Transgenic animals” carrying beta-globin gene and its promoter. Should be expressed in reticulocytes. But.. NO.

Question 19

Locus Control Regions

Answer 19

• In transgenes (expression vector with beta-globin gene and promoter injected into animal and incorporated into the genome of the recipient animal), presence of LCR resulted in:
• Tissue-specific transgene expression
• Genome integration-site independent expression
• Copy-number dependent expression ( increase in gene expression level as gene copies increase)
• Transgenes with enhancers often expressed in some animals and not in others– due to integration site.

Question 20

The Beta-Globin Locus Control Region (continued)

Answer 20

• The composition of enhanceosomes bound to the LCR varies at different developmental stages as do the proteins bound to the individual beta-like globin gene promoters.
• Result: appropriate “switches” in genes expressed during different stages in development.

Question 21

Yeast Enhancer and Silencer Sequences

Answer 21

• In the yeast, transcription of genes in the galactose utilization pathway is carefully regulated by enhancer-like sequences.
• When galactose is the only sugar available, wild-type yeast induce transcription of four enzyme-producing genes, GAL1, GAL2, GAL7, and GAL10
• Together these import and then break down galactose.
• How to turn on all four genes at the same time? Common enhancer associated with each.
• Induction of group of enzymes: you will be studying the bacterial lac operon.

Question 22

Using Galactose as an energy source in Yeast

Answer 22

• The enhancer element associated with each gene in this pathway is called the Upstream Activator Sequence (UASg)
• By the way, notice the direction of transcription of the four genes and the presence on more than one chromosome.

Question 23

Regulation of GAL gene transcription (example of inducible expression)

Answer 23

a. Galactose is absent
- Gal 4 and Gal 80 are made from two constitutively-active genes.
- The UASg includes two copies of 17 base pair Gal4 binding sequence
- Note: Gal4 is bound by Gal80 and is unable to bind UASg in the absence of Galactose.
- There is no transcription of the GAL genes.

Question 24

Gal 3 (constitutively expressed) binds galactose when galactose is present; this complex now binds Gal 80, causing Gal 80 to release Gal 4.

Answer 24

b. Galactose is present
- Gal4, Gal80, and Gal3 are constitutively expressed.
- Galactose is the inducer
- Gal 80 is bound by Gal3; Gal4 binds to UASg and activates transcription.

Question 25

Gal 4 Activation of Transcription

Answer 25

• Gal 4 binding of UASg leads to the formation of a multiprotein complex called the Mediator.
• The Mediator is an enhanceosome that induces formation of a DNA loop, making contact with the general transcription apparatus at the GAL gene promoters in cis. Mediator-like complex conserved among all eukaryotes.
• Transcription of GAL genes is dependent on transcription activation by Gal4/UASg binding and subsequent Mediator formation.

Question 26

Transcription Repression of the Yeast GAL1 gene (activation repression vs lack of activation)

Answer 26

• Lack of GAL1 expression is not due only to the inability of Gal 4 to bind UASg enhancer (when galactose is not present).
• In presence of glucose, GAL1 gene expression is actively repressed.
• Mig1 produced only when glucose is present in the cell.

Question 27

Insulator Action

Answer 27

• Silencer: directly represses gene expression (as in Mig1/Tup1 repression of Gal1 expression)
• Insulator: prohibits gene activation by an enhancer by BLOCKING enhancer/promoter interaction
• Enhancer activity help initiate transcription
• Insulator sequence blocks enhancer action and can redirect enhancer actively to another gene.
• A particular enhancer activates a gene in preference over a nearby enhancer whose action if blocked.
• Insulators may direct the formation of DNA loops that contain enhancers and the genes they activate.
• The insulator is not really a “sheet”. Cartoon of what is actually a protein complex.

Question 28

Chromatin Structure Regulates Gene Expression

Answer 28

• Heterochromatin: highly condensed, usually inactive transcriptionally. Darkly stained regions of chromosomes.
• Constitutive: condensed in all cells. (most of the Y chromosome and all pericentromeric regions)
• Facultative: condensed in only some cells and relaxed in other cells. (position effect variegation, X Chromosome in female mammals)
• Euchromatin: relaxed, usually active transcriptionally. Light stained regions of chromosomes.

Question 29

Heterochromatin: Position effect Variegation (PEV)

Answer 29

a. Wild type eye color
- w+ allele is expressed
b. Variegated eye color
- Inversion moves w+ near the centromere.
- Heterochromatin spread is variable.
- w+ allele is expressed in some spots and w+ is silenced in other spots.
- The eye is divided in specialized cells called ommatidia.

Question 30

Discovery that PEV was DUE to heterochromatin spreading:

Answer 30

• Some Su(var) mutations are caused by defective expression of heterochromatin protein-1 (HP-1)
• HP-1 is associated with centromeres, telomeres, and other heterochromatic regions in Drosophila.
• HP-1 is a nucleosome-binding protein that targets lysine in position 9 of histone H3 if it is methylated (H3-K9 methyl)- HP-1 binds this methyl group.
• Absence of HP-1 interferes with Heterochromatin formation and suppresses variegation (no way for heterochromatin to spread).

Question 31

Overview of Chromatin Remodeling and Chromatin Modification

Answer 31

• The defining feature of eukaryotic DNA (vs Bacterial) is its packing into chromatin
• Chromatin packing inhibits TATA-binding protein (TBP) and RNA Pol II access to promoters.
• Chromatin, therefore, serves to regulate expression of genes.
• Housekeeping genes (usually no TATA) have open promoters with a Nucleosome-Depleted Region.
• Most other genes are either tissue-specific or inducible and have conserved TATA box in their promoters. These promoter are closed.

Question 32

How to gain access to Closed Promoters?

Answer 32

• First, some regulatory sequences near TATA are not tightly bound by histones, so that regulatory proteins can easily gin access to those DNA sequences
• Second, DNA-binding pioneer transcription factors recruit proteins that change the chromatin:
• chromatin remodelers change the distribution or composition of histones.
• chromatin modifiers enzymatically modify histones by adding or removing acetyl or methyl groups to particular lysine residues.

Question 33

Chromatin Modification

Answer 33

• “Marks” on the amino-terminal tails of histones - usually on a lysine (K)
• Writers and erasers are recruited to chromatin by trans-acting transcription factors.

Question 34

Euchromatin and Heterochromatin

Answer 34

• Euchromatin becomes Heterochromatin when it goes through a Deacetylation Methylation reaction.
• Heterochromatin becomes Euchromatin when it goes through a Demethylation Acetylation reaction.

Question 35

Overview: Opening and Closing chromatin around a gene

Answer 35

• Histone acetyl-transferases (HAT) = writer

- Histone de-acetylases (HDAC) = eraser

Question 36

Modified histone N-terminal “tails” change strength of nucleosome interaction with DNA

Answer 36

• Positively-charged histone tails interact with negatively charged DNA
• Acetylation of Lysine in histone tails- removes the positive charge of Lysine– making tails less positively charged.

Question 37

Histone Methylation

Answer 37

• Methyl (CH3) groups are added to the N-terminal histone tails by histone methyltransferases (HMTs)= writers
• Lysine is frequently targeted for methylation
• Methylation plays a role in converting open chromatin to closed chromatin.
• Demethylation is carried out by histone demethylases (HDMTs) = erasers
• As already discussed, the methyl group (also true of acetyl groups) can be a binding site for other proteins that in turn, affect chromatin structure = readers.

Question 38

Chromatin Modification (cont)

Answer 38

“Histone code” = combination of histone modifications

- no one mark is sufficient to determine expression/repression.

Question 39

Chromosome 15 (note: not X-linked genes)

Answer 39

• On the maternal chromosome, an enhancer drives expression of H19 and an insulator protein blocks IGF2 expression. (OFF)
• On the paternal chromosome, methylation inactivates the ICR and blocks the H19 expression; the enhancer drives IGF2 expression. (ON)
• In this case, methylation is “closing” an insulator (not a promoter)

Question 40

Chromatin Structure helps us identify regulatory DNA sequences

Answer 40

• EX: DNAse 1 hypersensitive sites= regions that are free of nucleosomes (or have unstable nucleosomes) and, therefore are accessible by the enzyme DNAse 1.

Question 41

Chromatin changes are “Epigenetic” modifications

Answer 41

1. Epigenetic modifications do NOT alter DNA sequence- therefore, not “genetic” changes- not changes in the genetic material =DNA.
2. Instead, epigenetic modification patterns alter chromatin structure.
3. They are transmissible during cell division (liver cell divides- daughter cells still have the liver phenotype)
4. They are reversible (HDAC, HDMT =erasers)
5. They are directly associated with gene transcription.

Question 42

A Role for RNAs in Gene Regulation at transcriptional level

Answer 42

• Long non-coding RNAs (lncRNAs) = long RNAs that lack substantial open reading frames.
• An example of an lncRNA is Xist, which is involved in X-chromosome inactivation in eutherian mammals.

Question 43

Other RNA-mediated mechanisms controlling gene expression

Answer 43

• RNAi - RNA interference (broad term) caused by dsRNA
• MicroRNA (miRNA)= gene transcript that has a palindromic sequence allowing RNA to fold on itself and from dsRNA.
• Small, interfering RNA (siRNA)= dsRNA formed by bi-directional and overlapping transcription. ex: can involve transposons with terminal repeat sequences.

Question 44

Gene silencing by Double Stranded RNA

Answer 44

• RNAi silences gene expression transcriptionally, or post-transcriptionally
• Small regulatory RNAs can bind to mRNA targets by complementary base pairing.
• This can lead to destruction of the target mRNA or can block its translation.
• Some regulatory RNAs enter the nucleus and bind DNA to block transcription of the target gene.

Question 45

Dicer Protein

Answer 45

Cuts RNA into 21-25 base pairs

Question 46

RISC = RNA-induced silencing complex =multiprotein

Answer 46

RISC binds to siRNA or miRNA and then denatures RNA.

Question 47

Guide strand

Answer 47

The guide strand is the strand that was not denatured by RISC. The one that WAS denatured by RISC is called the Passenger strand.

Question 48

Sources of dsRNA

Answer 48

• dsRNA viruses infecting the cell
• Bi-directional transcription of DNA (e.g. repetitive sequences)
• Micro-RNA genes: RNA forms stem-loop structure.

Question 49

RNA-induced Transcriptional Silencing (RITS) in Yeast

Answer 49

Is responsible for heterochromatin formation at centromere.

Question 50

RNAi as a Research Tool

Answer 50

• RNAi can be used in a multitude of ways in research
• RNAi can be used to selectively “knock down” expression of selected genes to determine the effect on the phenotype.
• It may also be effective in medicine, where it might be used to control expression of genes that produces too much transcript, such as in cancer, or produce abnormal transcripts.