L9: Genome Editing & Mechanisms of Transcription Flashcards
(47 cards)
1
Q
DNA replication vs transcription - similarities
A
- 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
2
Q
DNA replication vs transcription - differences
A
- 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
3
Q
RNA Pol - prokaryotes vs eukaryotes
A
- prokaryotes: have a single one
- eukaryotes: have at least three (RNA Pol I-V)
4
Q
function of RNA Pol I
A
transcribes large rRNA genes
5
Q
function of RNA Pol II
A
transcribes protein encoding genes
6
Q
function of RNA Poll III
A
transcribes tRNA, 5S rRNA, and small nuclear RNA genes
7
Q
function of RNA Pol IV and V
A
- only found in plants
- transcribe small interfering RNAs involved in transcriptional silencing
8
Q
RNA Pol - prokaryotic RNA Pol and eukaryotic RNA Pol III
A
both drive mRNA expression and have an active center cleft
9
Q
RNA Pols - what is the active center cleft
A
- its a highly positively charged region of the enzyme
- can be occupies by negatively charged DNA
10
Q
RNA Pols - how does RNA Pol know where to start transcription?
A
- promoters
- where RNA Pols bind to
11
Q
transcription in prokaryotes - prokaryotic promoters
A
- 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
12
Q
transcription in prokaryotes: prokaryotic promoters - asymmetrical promoter sequences
A
- 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
13
Q
transcription in prokaryotes: prokaryotic promoters - consensus sequence
A
- 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
14
Q
transcription in prokaryotes - prokaryotic RNA Pol
A
- core polymerase
- holoenzyme
15
Q
transcription in prokaryotes: prokaryotic RNA Pol - core polymerase
A
- can initiate transcription at any point on the DNA molecule
- not what we want, want specificity
16
Q
transcription in prokaryotes: prokaryotic RNA Pol - holoenzyme
A
- core polymerase with initiation factor sigma (σ^70)
- initiates transcription only at promoter elements
- sigma binds specifically to -35 to -10 promoter elements
17
Q
transcription in prokaryotes - termination of transcription
A
- uses terminators
- sequences that trigger dissociation of RNA polymerase from DNA and the release of the RNA chain
18
Q
transcription in prokaryotes: termination of transcriptions - what are the two types?
A
- Rho-dependent terminators
- Rho-independent terminators
19
Q
transcription in prokaryotes: termination of transcriptions - Rho-dependent terminators
A
- 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
20
Q
transcription in prokaryotes: Rho-dependent terminators - method of termination?
A
- 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)
21
Q
transcription in prokaryotes: termination of transcriptions - Rho-independent terminators
A
- do not require protein factors
- consists of two sequence elements
22
Q
transcription in prokaryotes: Rho-independent terminators - sequence elements
A
- 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)
23
Q
transcription in eukaryotes
A
- gene expression is more complex compared to prokaryotes
- eukaryotes have differential gene expression
24
Q
transcription in eukaryotes - differential gene expression
A
- cells vary in the genes they express
- responsible for creating different cell types
- dictate the development of multicellular organisms
25
transcription in eukaryotes - promoter
DNA site where RNA Pol binds to initiate transcription
26
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
27
transcription in eukaryotes - regulatory sequences
- sections of DNA that control the activity of genes
- such as **enhancers** or **silencers**
28
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)
29
transcription in eukaryotes - RNA Pol II core promoter
- about 50bp long
- contains (1) TATA element, (2) TFIIB recognition elements (ex: BRE)
30
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
31
transcription initiation in eukaryotes - how is the pre-initiation complex involved in transcription?
1. initiated by **TBP**
2. **TAFs**
3. **TFIIB** enters the complex
4. **TFIIH** is recruited
5. ATPase activity and kinase activity
32
transcription initiation in eukaryotes: pre-initiation complex - TBP
- a TATA-Binding Protein
- recognizes the TATA element
33
transcription initiation in eukaryotes: pre-initiation complex - TAFs
- TBP-Associated Factors
- is brought in by TBP
34
transcription initiation in eukaryotes: pre-initiation complex - TFIIB
- enters pre-initiation complex after TBP
- bridges TBP and RNA Pol
35
transcription initiation in eukaryotes: pre-initiation complex - TFIIH
- recruited by
- functions as an ATPase and kinase
36
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
37
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**
38
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
39
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
40
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
41
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
42
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
43
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
44
transcription termination in eukaryotes - "torpedo model" of transcription termination
**Rat1** (in yeast) and **Xrn2** (in humans)
45
"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
46
"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
47
"torpedo model" of transcription termination: Rat1 and Xrn2 - what transcription termination in prokaryotes does this resemble?
Rho-dependent
