Transcription Regulation and Chromatin Remodeling

Question 1

Explain the importance of gene expression control in biological systems

Answer 1

Gene expression control
* The human genome has between 20,000 and 25,000 genes.
* 90% of the genes enxode proteins.–> mRNA
* The activities of proteins determine the phenotype of a person.
* Cells regulate protein activities in many different levels, such as transcription, phosphorylation, protein degregation and more.
* It is important to experimentally calculate the quantity of mRNA of a gene, the quantity of the protein of a gene, and the quantity of the active protein of a gene.
* How do the right genes get expressed in the right cells at the right time?

Transcription overview:
* RNA polymerases: Synthesize RNA in 5’ to 3’ direction by adding a NTP (like ATP, UTP..)
* No primer required
* RNA product has same sequence as coding strand, U in place of T

Transcription factor overview:
* Multiple transcription factors assemble at the promoter region
* Transcription does not occur without transcription factors
* Individual gene with unique promoter sequence, dictates which transcription factors are required

Types of transcription factors:
* General transcription factors: always present for mRNA synthesis
* Activators: recruited by enhancers to stimulate expression and recruit coactivaters–> speeds up rate of transcription
* Repressors: recruited by silencers to suppress expression–> slow transcription
* Mutliple transcription factors assemble at a promoter region
* Transcription does not occur without TF.
* Each has unique promoter region

Quantities:
* mRNA of a gene
* Protein of a gene
* Active protein of a gene

Question 2

List the various cellular processes that affect the protein activities in cells and their effects on the quantities of mRNA and proteins.

Answer 2

mRNA
* contains coding sequence for protein synthesis
* Synthesized from the transcription start site.
* Produced by the maturation of the initial transcript while being synthesized.
* G-cap and poly A tail- increase the half life of mRNA.
* Splicing- removes introns between exons

RNA Polymerase II
* Transcribes the protein-encoding genes to produce mRNA.
* Requires general transcription factors (TFIIA, TFIIB, TFIID TFIIE, TFIIF and TFIIH)
* C-terminal domain of the largest subunit of RNA Polymerase II
* Highly conserved YSPTSPS repeat
* Phosphorylated in RNA polymerase II that actively synthesizes RNA.
* Recruits various proteins for RNA processing.

Question 3

Identify the functions of various proteins involved in transcription and transcription regulation.

Answer 3

TFIID
* Binding of RNA polymerase II to a promoter requires the initial contact of the promoter with TFIID.
* Multi-subunit complex with TATA-binding protein (TBP) and different TBP-associated factors (TAFs)

TATA box
* A consensus sequence located 27 bp upstream of the transcription start site. More common for housekeeping genes.
* Recognized by the TATA binding protein.

Genes without TATA box
* An analogous AT rich region exists at the same location; TIID binding is still necessary.
* Other gene-specific TFs help TIID binding to the promoter

Question 4

Describe the role of transcriptional factors in transcriptional initiation.

Answer 4

Formation of the pre-initiation complex
* TFIID recruits TFIIA and TFIIB (DAB complex)
* DAB recruits RNA polymerase II and over 20 other general TFs

TFIIH
* Complete the pre-initiation complex with the histone arch that is stable
* Phosphorylates the C terminal domain CTD of RNA polymerase II
* Unwinds DNA
* Once phosphorylated, RNA polymerase II initiates RNA synthesis and leaves the pre-initiation complex–> elongation.

Activity of transcription factors are frequently regulated by binding to a signaling molecule or phosphorylation.
* Estrogen receptor is activated by binding to a signaling molecule or phosphorylation.

DNA sequences that bind regulated transcription factors are often referred to as response elements
* SRE- sterol response element
* CRE- cAMP response element

Question 5

List the types and functions of the modifications required for mRNA maturation

Answer 5

Alternative splicing increases the diverisity in proteins.
* Tropomysin-> critical protein for muscle contractions, Different muscle cells–> different splicing variants, Some exons skipped during splicing
* DNA methylation–> Cytosine in CpG is frequently methylated by DNA methyl transferase in human genome. CpG islands are commonly found in the promoter regions. Methylated CpG in the promoter region silences the transcription of the gene.

Chromatin remodeling:
Organization tactics:
* Heterochromatin- DNA tightly packed with nucleosomes, genes are repressed
* Euchromatin- DNA is less packed with nucleosomes, and genes are active

Covalent mods at N-terminal tails controls in part chromatin packing
* Acetylation on lysine → loss of one positive charge
* Phosphorylation on serine or threonine → addition of two negative charges
* Methylation on lysine or arginine → no change in charge

Question 6

Explain the mechanism of epigenetic controls of transcriptional regulation.

Answer 6

Epigenetics:
* heritable traits without the change in DNA sequence
* Parental experiences can be inherited by the children

DNA methylation
* Methylation of CpG islands
* Heritable; conserved during replication
* Regulates gene activities

Histone modifications
* Covalent modifications at the N-terminal tails
* Heritable; conserved during replication
* Regulates gene activities

Histone modifications are coordinated with DNA methylation.

Question 7

Describe the functions of the enzymes and the guide RNA in RNA interfrence and CRISPR-Cas9 gene editing.

Answer 7

RNAi:
* Double stranded RNA suppresses the translation or a protein by targeting mRNA
* Dicer cleaves the double stranded RNA to 21-23 nucleotide small interfering RNA
* The guide (antisense) strand is incorporated to the RNA-induced silencing complex
* RISC cleaves mRNA complimentary to the guide strand.
* The guide strand with imperfect complementarity miRNA still suppresses translation by forming micro-RNA-protein that suppresses the translation. (miRNP).
* Synthetic dsRNA is routinely used as a way to suppress the activity of a specific gene in labs.

CRISPR
Clustered regulatory interspaced short palindromic repeats (CRISPR)
* Found in bacteria and archaea
* COllection of DNA fragments of bacteriophage
* Used to detect and destroy DNA during subsequent infections

Cas9
* RNA-guided DNA endonuclease cleaves DNA complimentary to the guide RNA
* In genome editing, introduces double strand break at the location of interest in the genome.