Lecture 17: Transcription and Chromatin Flashcards
Protein activity
-determine phenotype of person
-regulated by cells in dif levels (transcription, tranaslation, phosphorylation, protein degradation, etc)
common experimental observations
-quantity of mRNA of a gene
-quantity of protein
-quantity of active protein
Human genome
~20,000 genes
-90% encode for proteins
-mRNAs (2% of total RNA)
-rest of the genes make rRNA, tRNA, regRNA
Human cell types
-200 kinds
-50-70% genes are active in cells
-majority for housekeeping
-each type expresses specific set of genes
RNA polymerases
-synthesize RNA in 5 to 3 direction
-add NTP complementary
-does NOT require a primer
-resulting RNA has same sequence as coding strand EXCEPT U instead of T
Transcription factors
-assemble at promoter region
-required for transcription
-type determined by unique promoter sequence
Transcription factor types
-general
-activators
-repressors
general transcription factors
alwys required for mRNA synthesis
Activators
-stimulate ecpression
-recruit coavtivators
-bind to ENHANCERS
repressors
-suppress expression
-bind to SILENCERS
mRNA
-coding sequence for protein synthesis
-made from transcription start site
-produced by maturation of initial transcript while being synthesized
G-cap and poly A tail
increase half life of mRNA
Splicing
removes introns between exons of mRNA
RNA polymerase II
-transcribe genes to produce mRNA
-requires general transcription factors (TFIID and TFIIH)
-C-terminal doman
C-terminal domain (CTD)
-largest subunit of RNA polymerase II
-highly conserved YSPTSPS repeat
-phosphorylated in RNA polymerase II that actively synthesizing RNA
-recruitsvarious proteins for RNA processing
TFIID
-required for binding of RNA polymerase II to a promoter
-multisubunit complex with TBP (tatabindingprotein) and TAFs (TBP associated factors)
TATA box
-27bp upstream of transcription start site
-more so in housekeeping genes
-recognized by TBP
Tata binding protein
-recognizes TATA box
Genes without TATA box
-analogous AT rich region exists at same loaction
-TIID binding is still needed
-other gene-specific TFs help TIID bind to promoter
pre-initiation complex
-TFIID recruits TFIIA and TFIIB (DAB complex)
-DAB recruits RNA polymerase II
-other general TFs associate
TFIIH
-completes pre-initiation complex
-phosphorylates CTD of RNA polymerase II (kinase activity)
-unwinds DNA (helicase)
Phosphorylation of RNA polymerase II initiates
RNA synthesis and leaves pre-initiation complex
=elongation
Regulation of transcription factors by:
-binding to signaling molecule
-phosphorylation
-response elements
response elements
-DNA seq that bind regulated TFs
-SRE and CRE
CRE
-cAMP response element
SRE
sterol response element
CREB
CRE binding protein
-activated by phosphorylation by PKA
Estrogen receptor
activated by binding to estrogen
Alternative mRNA splicing
-increases diversity in proteins
-human genome has only 20,000 protein-coding genes but make 80,000 proteins
-cells can generate multiple proteins from a single gene via alternate splicing
tropomyosin
-muscle contractions
-different musc cells produce dif kinds
-splicing skips some exons
DNA methylation
-Cytosine in CpG by DNA methyltransferase
-silences transcription of gene if occurs in promoter region
imprinting
methylation of CpG is restored after replication and inherited by daughter cells
Chromatin
-organizes DNA and positions nucleosomes
-regulates protein access for transcription
heterochromatin
-DNA tightly packed w nucleosomes
-genes repressed
Euchromatin
-DNA less packed
-genes active
Mods to Chromatin at N terminal tails
-controls packaging
-acetylation, phosphorylation, methylation
acetylation on lysine
loss of one positive charge
phosphorylation on serine or threonine
add 2 negative charges
methylation on lys or arg
no change in charge
-may activate or repress expression according to location
Histone code
-patterns of the covalent mods of histone
-determine chromatin structure
-recruit proteins responsible for remodeling and transcription
acetylation and phosphorylation
-reduce positive charges on histones
-bind less tightly to DNA
-produce euchromatin
-increase transcription
Epigenetics
-heritable traits without change to DNA sequence
-ex: parental experiences
DNA methylation and histone modifications (epigenetics)
-heritable
-conserved during replication
-regulates gene activities
Histone modifications
coordinated with DNA methylation
RNA interference (RNAi)
-ds RNA suppresses translation by targeting mRNA
-used in labs to supress genes
CRISPR
-clustered regulatory interspaced short palindromic repeats
-found in bacteria and archea
-collection of DNA fragments of bacteriophages
-detect and destroy DNA during infections
Cas9
-RNA guided DNA endonuclease
-cleaves DNA complimentary to the guide RNA
CRISPR-Cas9 genome editing
-expresses Cas9 and guide RNA in target cell
-introduces DSbreak at certain location
