L4-6: Regulation of Gene Expression Flashcards

Question

Molecular mechanism of offspring genetic diversity

Answer 1

1. Random assortment of chromosomes (meiosis), 2. Crossing over (chromosomes switch chunks of DNA) 3. Random fertilisation (chance alone is responsible for which sperm meets which egg)

Answer 2

tandem repeat sequence TTAGG

Answer 3

Telomeres maintain chromosome stability by protecting ends from exonuclease digestion or recombination and also ensure complete chromosome replication and proper segregation.

Answer 4

Reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle. When RNA primer is removed from the 5' end of the lagging strand, a strand of parent DNA remains unreplicated. Telomerase binds to the 3' overhang of ssDNA and ads dNT to the end of the parent DNA, complementary to its primer. Telomerase continues to move down the parent DNA, adding additional repeats. Primase, DNA polymerase and ligase synthesise the lagging strand in a 5'-3' direction

Answer 5

Chromatin is composed of nucleosomes (DNA wrapped around 8 histone proteins) and linker DNA. The histone protein H1 is located outside the nucleosome

Answer 6

11nm; ~147bp; 1.7 times

Answer 7

The histone octamer is composed of one H3/H4 tetramer and two H2A/H2B dimers: 1. H3 histone binds to H4 twice to form an H3/H4 tetramer (do not need DNA for binding) 2. DNA then interacts with the H3/H4 tetramer, after which the H2A/H2B dimers bind (H2A-H2B) — (H3-H4)x2 — (H2A-H2B) 3. H1 acts as glue to seal the nucleosome from the outside

Answer 8

``` Histone acetyltransferase (HAT) acetylates conserved lysine residues on histone proteins. Histone acetylation generally increases gene expression. In general, histone acetylation leads to increased transcription and gene expression through disruption of strong ionic interactions between positively charged amine groups on histone proteins and the negatively charged phosphate backbone of DNA. HATs acetylate specific lysine residues on histones, neutralising their charge and disrupting their ionic interactions with DNA. Acetylated lysines also act as interaction sites for other DNA-associated proteins. This causes loosening of chromatin structure; highly acetylated histones form more accessible chromatin, allowing transcriptional machinery access to the DNA. Acetylation is not always associated with enhanced transcriptional activity, e.g. acetylation of H4K12 has been associated with condensed and transcriptionally inactive heterochromatin. Histone deacetylases (HDAC) removes the acetyl group on acetylated histone lysine residues, restoring their positive charge and their ability to bind DNA, restoring the condensed chromatin structure. Deacetylation is associated with transcriptionally inactive heterochromatin. ```

Answer 9

Histone methyltransferases (HMT) methylate R groups of lysine and arginine residues on histone proteins Methylation of histones can either increase or decrease transcription of genes, depending on which amino acids are methylated and the number of methyl groups attached. Methylated histone tails serve to recruit other proteins and protein complex which regulate chromatin activation/inactivation Methylation events that weaken the interaction between histones and DNA are associated with increase in transcription, as they allow DNA to unwrap itself from histone proteins and for transcriptional factors to bind Methylated histones can either repress or activate transcription depending on the site of methylation e.g. dimethylation of lysine 9 on histone H3 (H3K9me3) in the promoter region of genes prevents excessive expression of these genes. In contrast, trimethylation of histone H3 at lysine 4 (H3K4me3) is associated with transcriptionally active euchromatin Histone demethylases (HDM) remove methyl groups

Answer 10

Hydrolysis of ATP to produce ADP can phosphorylate histone protein, creating negatively charged residues which won’t bind DNA Hydrolysis of ATP may also create enough energy to change conformation of protein, disrupting the interaction between histones and DNA

Answer 11

The CpG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5'→3' direction Methylation of cytosine to 5-methyl cytosine in mammalian DNA Catalysis by DNA methyltransferases (DNMTs) Site of methylation is the cytosine of a CpG doublet. This is a palindrome (i.e. present on both strands)

Answer 12

Basal transcription factors assist RNA polymerase in the recognition of promoter sequences and unwinding the DNA double helix, among other functions. Required for all transcription events.

Answer 13

- A subunit of TFIID, binds to the negatively charged phosphates of the TATA box through its positively charged lysine and arginine residues - TBP sits aside the TATA box as a molecular ‘saddle’, inducing a sharp bend in the DNA through insertion of four phenylalanine residues into the minor groove between base pairs, partially unwinding the helix, and doubly kinking it. - This initiates separation of the DNA strands, and is assisted through the weak interactions between adenine and thymine in this A-T rich region - This separation of the strands allows RNA polymerase II to begin transcription

Answer 14

TBP, a subunit of TFIID, bind the TATA box in the core promoter region TBP recruits TFIIA, followed by TFIIB to join initiation complex, stabilising the PIC TFIIB recruits TFIIF bound to RNA polymerase II TFIIE joins the complex, recruiting TFIIH

Answer 15

- Subunits of TFIIH with ATP-ase and helical activity create negative superhelical tension in the DNA, causing ~14 base pairs to unwind, and form a transcription bubble. - TFIIH protein complex hydrolyses ATP into ADP. - With the release of a free phosphate, CTD:Carboxyl Tail domain of Pol II is phosphorylated by TFIIH on serine residues 2, 5 and 7 - RNA polymerase II is moved downstream ~40 nucleotides

Answer 16

Early after transcription initiation, the ternary elongation complex (consisting of RNA pol II, DNA template and RNA transcript) is arrested at a “checkpoint” to ensure proper pre-mRNA capping. In eukaryotes, upon promoter clearance, Kin28 subunit of TFIIH phosphorylates serine 5 on the CTD of RNA polymerase II, leading to the recruitment of capping enzyme (CE) and the nascent RNA becomes capped during this first stage of elongation. The RNA cap is formed by addition of a methylated guanosine to the 5' end of the RNA through the action of capping enzyme and an RNA (guanine-7) methyltransferase. Release from this checkpoint involves the kinase action of P-TEFb, which targets Ser 2 on the CTD. Subsequently during elongation, phosphatases such as PTase dephosphorylate Ser 5 residues within the CTD; Ser 5-specific phosphatase Ssu72 is involved in allowing the correct transcript cleavage necessary for efficient termination. Both Ssu72 and the FCP1 phosphatase, which also targets Ser5, are involved in recycling the RNAP II for re-initiation and subsequent rounds of transcription, although the specifics of recycling are currently unknown.

Answer 17

Protects transcript from degradation and helps with initiation of translation After the initial pre-mRNA transcript has been created the first major modification is the addition of a 5' methyl guanosine cap. Addition of the 5' 7-MG cap is important for two reasons: the 5' caps are recognized by protein factors that initiate translation, and it also helps protect the transcript from nucleases.

Answer 18

Once RNA polymerase reaches the terminator region, it releases the capped pre-mRNA molecule Cleavage factors bind to specific regions in the pre-mRNA segment (GU-rich region and AAUAAA), the 3’ end is reconfigured and stabilising factors bind Poly-A polymerase binds and the pre-mRNA molecule is cleaved, disassociating the complex Poly-A polymerase then synthesises the poly-A tail by adding adenines to the cleavage site

Answer 19

cis-regulatory elements (CREs) that enhance transcription of genes on the same molecule of DNA.

Answer 20

TFs/trans-elements that bind enhancers or promoter-proximal regions to increase gene transcription

Answer 21

TFs that bind to silencer region, occupy region where TFs bind

Answer 22

Silencers are CREs that can bind transcription regulation factors called repressors, thereby preventing transcription of a gene.

Answer 23

Chromatin Immunoprecipitation (ChIP)

Answer 24

ChIP aims to define the cistrome - the set of cis-acting targets of trans-acting factors - and determine whether specific proteins such as TFs are associated with specific genomic regions, such as promoters or other DNA binding sites ChIP also aims to determine the specific genomic location of histone modifications DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody. The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.

Answer 25

Technique used to identify promoters/TF-binding regions Specific point mutations or deletions are made in specific regions of the promoter and the transcription of the gene is measured; the level of transcription can be attributed to the contribution of the region of the promoter The ability of upstream promoters can be easily assayed by removing segments from the 5' end, and the same for the 3' end of the strand for downstream promoters. Procedure: Region of DNA thought to act as promoter is cloned and extracted; this ensures promoter is the only factor affecting expression Region is sequenced to identify differences in WT and mutated promoters Region is digested with appropriate nucleases to systematically remove elements of the region Gene expression levels are measured through a reporter gene ligated to the promoter region

Answer 26

Oligo-dT purifications of mRNA will result in extraction of mRNA transcripts with polyA tails only; Only mRNAss contain a poly-adenylated tail.

Answer 27

1. Oligo dT primer + reverse transcriptase, dNTPs | 2. Treat with NaOH to destroy RNA

Answer 28

A transcription factor is split into its constituents elements: DNA binding domain (DBD), specific to the TF, and an Activation Domain (AD) that is generic across TF. The DBD is fused to a protein, termed bait. The AD is fused to a different protein, termed prey. In the yeast two hybrid system, two plasmids are transformed into yeast, one plasmid containing the sequence for the DBD and bait protein, as well as a selectable marker such as a gene coding for Leucine production. The other plasmid encodes for the AD, prey and a different selectable marker such as tryptophan. The two plasmids are inoculated and transformed into a yeast strain deficient in both of the selectable markers and depends upon them for growth. This mixture is then plated and incubated on a medium deficient in both selectable markers, thereby selecting for transformants with both plasmids only. Within the yeast, the DBD-domain will bind to its target sequence, a reporter gene such as Luc or GFP. If the prey and bait proteins bind, their interaction will bring the AD and BDB in close proximity and transcription will occur, and the reporter gene will be transcribed and translated.

Answer 29

If gene of interest is integrated in transcriptionally inactive heterochromatin, it will not be transcribed Genome editing for targeted transgene integration requires creating a double-stranded break in the target sequence to increase efficiency of homological recombination as well as flanking (to 0.001%) as well as adding flanking regions to your cassette identical to the flanking regions of the target sequence.

L4-6: Regulation of Gene Expression Flashcards

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