BIOC 3 Flashcards
(28 cards)
gene expression
the process by which information from a gene is used in the synthesis of a functional product.
why is gene expression important?
cells are made of and function - product of gene expression
products of gene expression: STRUCTURE: -cytoskeleton -membranes -cell wall BIOCHEMICAL REACTIONS: -catabolism =breaking down molecules- release energy -anabolism =building of bigger molecules
CELLULAR AND INTRACELLULAR COMMUNICATION
GENE EXPRESSION
control of gene expression
DEVELOPMENT:
can be altered when gene expression is not properly controlled (mutant phenotype)
ENVIRONMENT:
need to respond to the environment
points of controls gene expression in eukaryotes
controlled at:
TRANSCRIPTION:
- which genes are copied- into mRNA
- measure using RT-qPCR + RNA-seq
mRNA PROCESSING:
-alternative splicing giving different forms- protein
POST-TRANSCRIPTIONAL:
- mRNA stability + translation efficiency (miRNA)
- determine how much protein - made from each mRNA
POST- TRANSLATIONAL
- covalent modifications - alter proteins function
- localisation
- degradation
transcription
process of creating a complementary RNA copy of a sequence of DNA
catalysed by enzyme DNA dependent RNA polymerase (RNA pol)
- adds rib nucleotides to 3- growing chain
- uses rib nucleotide triphosphates (NTP: ATP,UTP ) as substrates
transcription in eukaryotes
- occurs in multiple places
- multiple types of RNA
- multiple DNA dependent RNA polymerase
RNA polymerase 11 (multi protein)
has multiple subunits - Rpb#
catalytic subunits related to subunits of prokaryotic RNA pol
additional subunits involved in regulation, pre-mRNA processing and transcription through chromatin
RNA polymerase 11 function
Transcription: 1: CLEFT -DNA enters and is unwound 2: CLAMP -closed when active -open when inactive 3:WALL -bends template strand into active site 4:PORE -NTPS enters active site - through funnel 5: ACTIVE SITE/CATALYTIC SITE -template base exposed to NTPs(pore) -NTP selected by base pairing -conformation changes =catalysis - added nucleotide 3' end of RNA 6:BRIDGE -alternates between bent/straight -pushed paired nucleotides out of active site 7:RUDDER -separates DNA +RNA (EXIT channel)
PROOF READING: -mistake=mismatch in DNA/RNA hybrid helix -decrease stability -pol ii back tracks (wrong direction) -RNA 3' end - goes into PORE -detected stimulates active site to become nuclease =cuts DNA -removes wrong base -3'end RNA -active site -TRNACRIPTION RESUMES
RNA polymerase 11 c-terminal domain (CTD)
c-terminal domain of the Rpb1 subunit
- not catalytic
- heptapeptide repeat (multiple copies of the same amino acid)
the CTD is reversibly phosphorylated- during transcription - kinase added and phosphatase removed
occurs on SER 2 and SER 5
alternation with other proteins
stage of transcription
1- pol 11 recruitment
2-initiaon and early elongation
3-productive elongation
4-termination
RNA pol 11- NEEDS HELP
- cant bind on its own
- cant recognise - START of genes on its own
- cant bind/transcribe -DNA wrapped in nucleosome
structure of protein-coding gene
CORE PROMOTER:
- DNA sequence - general transcription factors bind+ recruit RNA pol 11
- close to TSS
- Small
PROMOTER:
- entire regulatory region around TSS
- includes core promoter
- upstream DNA facilitates the transcription of a particular gene.
- larger DNA
CIS - REGULATORY ELEMENT (CRE):
- short DNA sequence - regulation of transcription
- cis=same molecule
histone tail modification
the amino n terminal region- histone monomers extend beyond the nucleosome = accessible for covalent modification.
ACETYLATION(ac) -added by histone acetyltransferases (HATS) and is removed by deacetylases (HDACs). -lysine residues -removed positive charge recognised by specific protein
METHYLATION(me)
- added by histone methyltransferases (HMTase) and removed by demethylases.
- lysine and arginine residues
- one ,two ,three groups added
- recognised by specific protein
nucleosome/chromatin remodelers
- ATP dependent
- disrupts interactions between DNA +histones
- side/eject (fully or partially) nucleosome -expose DNA region
- maintain packaging-makes DNA accessible to transcriptional machinery + DNA binding proteins
- work in conjunction with HISTONE CHAPERONES
activator- dependent recruitment of RNA pol 11
process:
1- promoter selection determined- by interactions of 1 or more transcriptional activators- with specific sequence near target genes
activators recruit components - transactional machinery to these genes through protein -protein interactions.
2-actiavtion of gene expression is induced - by the sequential recruitment of large subunit protein co-activator complexes through binding to activators.
activators also recruit ATP-dependent nucleosome-remodelling complexes- which move or displace histones at the core promoter, facilitating -rapid recruitment + assembly of co-activators and the general transcriptional machinery.
3- together- co-activator and nucleosome remodels facilitate the rapid recruitment of RNA pol 11 and the general transcriptional factors (GTFS) TF11A, TF11B,TF11D,TF11F and TF11g to form the pre-imitation complex (PIC) on the core promoter.
activator-dependent recruitment
Cis-regulatory element:
recognised + bound by DNA binding proteins
-enhancer promoters transcription- BOUND by activator
-silencer inhibits transcription- BOUND by repressor
Activators: -recognise + bind a CRE based on DNA sequence (target specific genes) -transcription factors -calledACTIATOR= 2 domains 1) activation= trans acting factors ( different) -TYPICAL domain 2)DNA binding domain both act independently
activation domain
protein-protein interactions:
- other activators
- co-activators
- not conserved
DNA binding domain
recognises a cis-regulatory element
- hold activator domain in the VICINITY of the promoter -
- used to classify activators-into families
- conserved
co-activators (protein complexes)
DIVERSE ROLES:
-Protein- protein interactions
-range of activators
general transcription factors + RNA pol 11
CHROMATIN MODIFICATION:
- HAT activity (histone H3/H4 acetylation)
- other histone tail modification
Chromatin remodelling at core promoter
CO_ACTIVATORS:
- histone H3/H4 acetylation
- other histone tail modifications
nucleosome remodelers recruited by activators +acetylated histone H3/H4
-slide/eject nucleosome from core promoter
RESULT= core promoter is accessible to RNA pol 11
TFIID recruitment to core promoter
1) binding core promoter elements
- different elements bound by different TFIID subunits
- most yeast have - least 1 element
- most mammalian genes (70%) - don’t have element but have multiple TSS (50-200bp)
2) protein-protein interactions- with co-activators and activators
3) histone tail modification
- H3/H4 acetylation, methylation of specific residues
pre-mRNA processing occurs during transcription
- addition of 5’cap
- introns spliced out
- addition of 3’ poly A tail
addition of 5’m7G cap to mRNA transcripts
1- UNCAPPED mRNA:
- 5’ triphosphate will be degraded by nucleases
- unstable transcript
2-mRNA CAPPING:
- capping enzyme (CE) recruited by ser 5 p of CTD
- triphosphatase
addition of 5’m7G cap to mRNA transcripts
1- UNCAPPED mRNA:
- 5’ triphosphate will be degraded by nucleases
- unstable transcript
2-mRNA CAPPING:
- capping enzyme (CE) recruited by ser 5 p of CTD
- triphosphatase `
