L9: Genome Editing & Mechanisms of Transcription Flashcards
DNA replication vs transcription - similarities
- polymerase synthesize new strand of nucleic acid complementary to DNA template
- involves opening and unwinding of DNA helix
- synthesis is always in 5’-to-3’ direction
DNA replication vs transcription - differences
- DNA Pol or RNA Pol (latter doesn’t need a primer)
- dNTPs (deoxyribonucleotides) or NTPs (ribonucleotides)
- only one DNA strand acts as template in transcription
- transcription product strand is displaced from template
- only fraction of genome is transcribed at a given time but the entire genome is replaced
- DNA replication is more accurate since it has more proof-reading mechanisms
RNA Pol - prokaryotes vs eukaryotes
- prokaryotes: have a single one
- eukaryotes: have at least three (RNA Pol I-V)
function of RNA Pol I
transcribes large rRNA genes
function of RNA Pol II
transcribes protein encoding genes
function of RNA Poll III
transcribes tRNA, 5S rRNA, and small nuclear RNA genes
function of RNA Pol IV and V
- only found in plants
- transcribe small interfering RNAs involved in transcriptional silencing
RNA Pol - prokaryotic RNA Pol and eukaryotic RNA Pol III
both drive mRNA expression and have an active center cleft
RNA Pols - what is the active center cleft
- its a highly positively charged region of the enzyme
- can be occupies by negatively charged DNA
RNA Pols - how does RNA Pol know where to start transcription?
- promoters
- where RNA Pols bind to
transcription in prokaryotes - prokaryotic promoters
- has two conserved regions: -35 element and -10 element (relative to transcriptional start site) separated by about 17bp
- promoter sequences are asymmetrical
- contain a consensus sequence
transcription in prokaryotes: prokaryotic promoters - asymmetrical promoter sequences
- bc they are asymmetric, they are directional (RNA Pol binds in one direction)
- the template strand is thus determined by the orientation of the promoter
transcription in prokaryotes: prokaryotic promoters - consensus sequence
- promoters closest to the consensus sequence are “stronger” and produce higher amounts of transcripts
- binds to sigma most effectively
- -35 sequence: T T G A C A
- -10 sequence: T A T A A T
transcription in prokaryotes - prokaryotic RNA Pol
- core polymerase
- holoenzyme
transcription in prokaryotes: prokaryotic RNA Pol - core polymerase
- can initiate transcription at any point on the DNA molecule
- not what we want, want specificity
transcription in prokaryotes: prokaryotic RNA Pol - holoenzyme
- core polymerase with initiation factor sigma (σ^70)
- initiates transcription only at promoter elements
- sigma binds specifically to -35 to -10 promoter elements
transcription in prokaryotes - termination of transcription
- uses terminators
- sequences that trigger dissociation of RNA polymerase from DNA and the release of the RNA chain
transcription in prokaryotes: termination of transcriptions - what are the two types?
- Rho-dependent terminators
- Rho-independent terminators
transcription in prokaryotes: termination of transcriptions - Rho-dependent terminators
- Rho: ring-shaped protein with six identical subunits
- binds to RNA as it leaves RNA Pol
- has ATPase activity that is used to terminate transcription
transcription in prokaryotes: Rho-dependent terminators - method of termination?
- Rho attaches to recognition site on RNA
- Rho moves along RNA, following RNA Pol
- RNA Pol pauses at terminator and Rho catches up
- Rho unwinds DNA-RNA hybrid in transcription bubble
- RNA Pol, rho, and RNA are released (termination)
transcription in prokaryotes: termination of transcriptions - Rho-independent terminators
- do not require protein factors
- consists of two sequence elements
transcription in prokaryotes: Rho-independent terminators - sequence elements
- nts = nucleotide
- 20nt inverted repeat: forms a hairloop which disrupts elongation
- 8 U nts: short stretch which holds RNA transcript to template and weak binding allows dissociation of the transcript (termination)
transcription in eukaryotes
- gene expression is more complex compared to prokaryotes
- eukaryotes have differential gene expression
transcription in eukaryotes - differential gene expression
- cells vary in the genes they express
- responsible for creating different cell types
- dictate the development of multicellular organisms
transcription in eukaryotes - promoter
DNA site where RNA Pol binds to initiate transcription
transcription in eukaryotes - general transcription factor (GTF)
- serve as initiation factors (perform role of σ)
- required for transcription
- expressed in all cells
- bc of this are not reflective and do not confer differential gene expression
transcription in eukaryotes - regulatory sequences
- sections of DNA that control the activity of genes
- such as enhancers or silencers
transcription in eukaryotes - regulatory transcription factor
- cell-specific factors that bind to the regulatory sequence
- dictates differential gene expression (only cells that have the regulatory protein will express the gene)
transcription in eukaryotes - RNA Pol II core promoter
- about 50bp long
- contains (1) TATA element, (2) TFIIB recognition elements (ex: BRE)
transcription initiation in eukaryotes - what is the pre-initiation complex
complete set of GTFs and RNA Pol II bound at the promoter and poised for transcription initiation
transcription initiation in eukaryotes - how is the pre-initiation complex involved in transcription?
- initiated by TBP
- TAFs
- TFIIB enters the complex
- TFIIH is recruited
- ATPase activity and kinase activity
transcription initiation in eukaryotes: pre-initiation complex - TBP
- a TATA-Binding Protein
- recognizes the TATA element
transcription initiation in eukaryotes: pre-initiation complex - TAFs
- TBP-Associated Factors
- is brought in by TBP
transcription initiation in eukaryotes: pre-initiation complex - TFIIB
- enters pre-initiation complex after TBP
- bridges TBP and RNA Pol
transcription initiation in eukaryotes: pre-initiation complex - TFIIH
- recruited by
- functions as an ATPase and kinase
transcription initiation in eukaryotes: pre-initiation complex - ATPase and kinase activity
- ATPase activity melts DNA
- kinase activity (adds a phosphate) frees RNA Pol from pre-initiation complex
transcription initiation in eukaryotes - “promoter escape” by RNA Pol
- RNA Pol must leave pre-inanition complex to synthesize DNA
- escape requires phosphorylation of Carbon-Terminal Domain (CTD) or “tail” of RNA Pol II via TFIIH
transcription initiation in eukaryotes: “promoter escape” by RNA Pol - CTD of RNA Pol
- CTD contains series of repeats (25-50) which serves as a target site for kinase
- phosphorylation of CTD facilitates “shedding” of GTF’s and release from the promoter
transcription initiation in eukaryotes - RNA 5’ and 3’ end-processing
- prokaryotes do not do this
- steps:
1. 5’ cap addition
2. polyadenylation - these two increase the stability of the transcript so it doesn’t get degraded by enzymes
transcription initiation in eukaryotes: RNA 5’ and 3’ end-processing - 5’ cap addition
- addition of a modified guanine base to the 5’ end of the transcript
- displays unusual 5’-5’ linkage involving three phosphate groups
transcription initiation in eukaryotes: RNA 5’ and 3’ end-processing - polyadenylation
- a specific sequence (polyA signals/tails) at the 3’ end of transcript will recruit polyadenylation enzymes
- RNA Pol II CTD carries two proteins to cleave the transcript: CPSF and CSTF
RNA 5’ and 3’ end-processing: polyadenylation - CPSF and CSTF
- CPSF: cleavage and polyadenylation specificty factor
- CSTF: cleavage stimulation factor. It binds to the polyA signal and cleaves transcript
RNA 5’ and 3’ end-processing: polyadenylation - what happens after CTD cleaves transcript
- Poly-A Polymerase (PAP) adds about 200 adenines to the cleaved end
- PAP works like an RNA Pol but does not require a template
transcription termination in eukaryotes - “torpedo model” of transcription termination
Rat1 (in yeast) and Xrn2 (in humans)
“torpedo model” of transcription termination - Rat1 and Xrn2
- they are RNases that recognize that RNA is still being made by RNA Pol II following cleavage (since its uncapped)
- they will quickly degrade RNA in a 5’-3’ direction until it catches RNA Pol II and triggers its release
“torpedo model” of transcription termination: Rat1 and Xrn2 - how does it know to cleavage the garbage strand vs productive RNA?
5’ cap and polyA tail prevents degradation
“torpedo model” of transcription termination: Rat1 and Xrn2 - what transcription termination in prokaryotes does this resemble?
Rho-dependent
