50 terms

Eukaryotic Transcription


Terms in this set (...)

General Considerations
-more complex than prokaryotes
-multicellular: use differential transcriptional regulation to establish different cell types
-critical to establish diversity of multicellular organisms
-certain genes are expressed in all cell types: housekeeping genes- routine metabolic function
-some genes are expressed in response to a particular stimulus, ex hormones
-some genes are expressed transiently or continuously in subsets of cells
-altered in a disease state: can be detected by measuring expression levels of genes and/or proteins in body fluids like blood and saliva
-relies on promotors
Promoters Definition
-DNA sequence that specifies the start of transcription
-recruits RNA pol
Transcription factors Definition
bind to DNA sequences and recruit RNA pol
-packaging of eukaryotic DNA
-DNA wrapped around histones
-must be open/close to access
Enhancers/ Silencers
other transcriptional control elements bind to activator and repressor chromatin remodeling factors
Chromatin Remodeling
1. Histone tail modification
2. DNA modification
Histone tail modification
histones have flexible tails that can be modified
DNA modification
-methylated on C and A
-on C and A
-methylation of CG (also called CpG) in promoters is associated with transcriptional repression
Covalent Modification of Histones or DNA
Writer Ez
add modifications to DNA/histones--> promote modification
Eraser Ez
remove modifications from DNA/histones
Reader Ez
recognize modifications on DNA/histones
-typically located immediately upstream of the transcriptional start site: 100-1000 bp
-access of regions mediated by enhancer/silencer genes
-far from promoter: adding enhancer to gene is not easy
-these act/inhib promoters by alternating chromatin state
-critical for initiating transcription
-often facilitate DNA looping, how access promoter region --> allows access for chromatin remodeling
-often facilitate DNA looping and function to make promoters accessible/inaccessible by directing the alteration of chromatin
-often bound by TF are called activators (2 domains)
-2 conserved domains
1. DNA-binding domain at the enhancer
2. RNA pol binding (or other protein factor-binding) domain that facilitates looping of DNA
-regulators of chromatin
-required to recruit RNA pol to DNA, when pol bound to DNA sigma factors recognize promoters
-binds to specific DNA sequences
-larger number in eukaryotes: 2,600 in humans (diverse and different from prokaryotes)
-usually respond to some kind of signal
-2 domains: DNA binding domain (DBD) and transcription activating domain
DNA binding domain (DBD)
-in TF, but usually signal sensing domain in TF that responds to signal/change in microbiome
Transcription activating domain
-in TF, mediated binding to other transcription regulation proteins that modify chromatin or modify the recruitment of RNA pol
3 types of RNA pols
RNA pol I, RNA pol II, RNA pol III
RNA pol I
-synthesizes rRNA, but not 5S rRNA
-unique among them, transcribes only one set of genes (rRNA) and only has to recognize single promoter structure
- DO NOT require TATA box in promoter: relies on distinct upstream control element (UCE)--> recruits specialized proteins
RNA pol II
-synthesizes mRNA and most snRNA and microRNA
-divided into core region: often contains TATA box
-regulatory regions highly varied in structure: reflecting the highly varied synthesis patterns of cellular proteins and the need for exquisite and complex regulation of these patterns
-synthesizes tRNA, 5S rRNA and other small RNAs
-promoters are more varied in structure than the uniform RNA pol I promoters- but not as diverse as RNA pol II promoters
-some promoters rely on TATA box- but many don't
TATA box
-minimal region capable of directing transcription in vitro and regulatory region
-important in recruitment and activation of RNA pol
Basal Transcription Complex
-role of TF's is to recruit the BTC to initiate transcription
-recognizes TATA box, relatively common promoter found about 30 bp upstream of transcription start site
-generally found in genes that exhibit noisy expression and thought to amplify expression
-TBP is general TF that binds to TATA box
-TFIID binds to TBP followed by other TF's
-RNA pol can then recognize the multi-protein complex and bind with other TF= pre-initiation complex
-TFIIH part of pre-initiation complex
-Transcription initiated
Transcription Binding Protein
TBP, general transcription factor that binds TATA box
TF, binds to TBP followed by other general TF's
Pre-initiation complex
-RNA pol can recognize basal transcription complex, bind to it, and with many other TF's
-one component is TFIIH
-Transcription initiated and then pol moves along DNA strand
phosphorylates RNA pol II triggering transcriptional activation
Factors to stimulate basal complex
-additional regions of DNA often recruit stimulatory factors that help control rate of transcription: CAAT box and G/C box
-genes with CAAT box seem to require it for the gene to be transcribed in sufficient quantities
CAAT box
-helps control rate of transcription
-upstream by 60-100 bases to initial transcription site
G/C box
-helps control rate of transcription
-upstream about 100-110 bases from transcription site
RNA pol I vs RNA pol III
RNA pol I:
-pre-intiation complex involves action of DNA-binding factor and promoter- selevtive factor --> recruits TBP and other factors that activate RNA pol I

RNA pol III:
-transcribed genes have internal promoters in transcribed region that recruit TBPand activating factors which recruit and activate RNA pol III
Internal Promoters
in transcribed region, recognized by large complex that recruit TBP
-in bubble of unwound DNA, where RNA pol uses 1 strand of DNA as a template to catalyze the synthesis of new RNA strand in 5' to 3' direction
-when only one RNA pol molecule occupies a promoter at a time, elongation involves multiple RNAs moving one after another along same DNA duplex
-transcription machinery needs to move histones out of the way every time it encounters a nucleosome, ATP dependent Ez can do this
-involves proofreading mechanism: replaces incorrectly incorporated bases, but doesn't function as well as DNA pol proofreading activity
Transcriptional Pausing
-RNA transcribed at a heterogeneous rate (even from same promoter)- in eukaryotes and prokaryotes
-not continuous--> interrupted by pausing events
-Pausing can include: halting transcription, sometimes involved backtracking events
-during backtracking the catalytic site becomes disengaged by 3' end of RNA--> rendering RNA pol inactive but stable
-pausing plays important roles in coordinating translation and transcription in Prokaryotes
-role in maintaining transcriptional fidelity, proofreading
-roles in overcoming roadblocks like nucleosomes
-can fine tune transcription by mediating premature termination
Termination of RNA pol I
-pre-mRNA is transcribed and a complex of proteins (FACTOR DEPENDENT) is recruited into termination site--> leading to termination and subsequent cleavage
-several specific termination sequences upstream of pause site, factors move on RNA and catch up to termination sequence
-similar to rho-dependent mechanism in prokaryotes
Termination of RNA pol II
-terminates transcription at random locations past the end of the gene being transcribed
-2 protein complexes (FACTOR DEPENDENT) recognize poly-A sequence leading to cleavage of RNA and resulting in poly A tail
-template independent addition of As as its new 3' end --> polyadenylation--> stabilizes RNA
-non polyadenylated RNA transcripts are rapidly degraded
Termination of RNA pol III (5S rRNA, tRNA, and other small RNA's
-terminates transcription in response to specific termination sequences in newly synthesized RNA
-can terminated efficiently without additional factors (FACTOR INDEPENDENT)
-often RNA that forms a hairpin followed by poly-U sequence, similar to rho-independent in prokaryotes
General Eukaryotic mRNA Processing
-active mRNA from pre-mRNA which will be modified
-pre-mRNA makes up hnRNA (heterogenous nuclear)
-in nucleus
-some required for transport to cytoplasm
-increase half-life
-include: 5' cap, poly A tail, splicing intron, cleavage/addition/ribose-base alterations like prokaryotes
5' Capping
-end of 5' RNA has free triphosphate group
-last phosphate removed by RNA triphosphatase
-cap added by Guanyl transferas: catalyzes reaction between 5' need of transport and GTP
-GTP added in inverted orientation --> 5'-5' triphosphate linkage
-guanosine methylated on 7 position after caping --> 7-methylguanylate cap (m7G)
-cap binding complex: cap binds 2 proteins in nucleus that forms CPC
-important for: nuclear export, translation, stability of mRNA (protects from degradation)
3' poly A tail
-begins as transcription terminates
-most sites contain AAUAAA
-3' most segment of new mRNA cleaved off by set of proteins that synthesize poly A tail at 3' end
-tail acts as binding site for poly A binding protein
-alt polyadenylation can occur in transcripts: influence stability of translation of transcript
Poly A binding protein
Promotes export from the nucleus, translation, and inhibits degradation
Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins.
-mutations at splice site common in disease
-splice do not, splice acceptor, branch site
Alternative splicing
allows multiple mRNAs- produce different proteins to be made from single transcript
Splice Donor
5' end of intron, exon G-U
Splice Acceptor
3' end of intron, A-G exon
Branch Site
-in intron, 30 nucleotides upstream of splice acceptor
-AT rich region with at least one A
Spliceosome Events
1. splice donor site cleaved
2. attaches to branch site to form lariat or loop structure
3. splice acceptor site is cleaved
4. intron degrades, 2 exon are ligated
-large RNA-protein complex made of 5 snRNPs
-assembly and activity of the spliceosome occurs during transcription of pre-mRNA IN NUCLEUS
-RNA components of snRNPs interact with intron and involved in catalysis