DNA to RNA synthesis and Processing part 1 Flashcards
What are the steps involved in transcription?
Initiation: recruitment of RNA polymerase
Elongation: promoter clearance and extension
Termination: release of RNA polymerase
What are the steps in Eukaryote transcription?
- proper transcription by RNA polymerase II requires co-factors
Mediator and co-mediator
Specific transcription factore (ie SOI, GAG,A HSF)
Transcriptional activators (often domains of TF’s)
Chromatin remodeling complexes (such as SWI/SNF, NURF)
Elongation factors
Termination Factors
n Eukaryotes: RNA pol II (mRNA) requires co-factors mediatore co-mediators, general TF, Chromatin remodeling, complexes, elongation factors, termination factors
Transcription basics
- RNA produced: complementary to template strand
matches sequence in DNA coding strand
The function of RNA polymerase is to copy one strand of duplex DNA into RNA
RNA produced is complementary to the template strand matching the sequence of the coding strand
What is the role of RNA olymerase?
RNA polymerase:
adds NTP’s 5’-3’ direction at 40/sec
does not need 3’ OH primer to start
binds dsDNA at promoter sites upstream of transciption start site
may require additional proteins to aid in binding and stability
overview of transcription
a transcription unit is a sequence of DnA transcribed into a single RNA starting at the promoter and ending at the terminator
Transcription unit defined by the presence of a promoter region, a coding region and a terminator region.
not that +1 indicates the actual transcription star
definition of upstream and downstream
the transcription process
- works 5’ - 3’
- DNA is temprarily denatured (10-20 bases at a time) typically 18 5. bp
- RNA is hybridized at 3’ end over a few bases (less than 12 bp)
- Formation of a transcription bubble
- RNA held in position by RNA-DNA hybrid formation over only a few bp
describe bacterial RNA polymerase holoenzyme
- 5 subunits
- alpha (x2) = required to assemble core protein
binds some activators promoter recognition (40KD) - Beta + Beta” = catalytic core (`55 NS 160 KD
- sigma = binds 10 and 35 promoter sites
increases specific binding is dispensable for polymerase (32-90 KD)
Bacterial RNA pol (only one) 5 SU
2 alpha SU responsible for promoter recognition and binding of activators
Catalytic SU are beta and beta prime (respectively 155 and 160KD)
Sigma SU binds at position -10 and -35 increasing specificity of binding at the proper location (-10 and -35 positions are important sequences)
Explain how RNA polymerase has a channel for DNA
The template strand is in red
Non-template strand is in yellow
DNA clamped between beta and beta’ SU.
DNA melted and formation of RNA transcript (brownish-gold)
Alpha 2 + sigman on opposite side of polymerase (grey and green)
Describe RNA polymerase and the unwinding of DNA
The ~10 and ~35 boxes
RNA polymerasa unwinds DNA ~10 bp upstream from transcription start
~10 box of Pribnow box: corresponds to the TATA box (consensus sequence is TATAAT)
~35 box: consensus sequence TTGACA
note the position +1 where transcrition starts (addition of nucleotides)
TTGACA Promoter ~35 bp upstream of start has a role in stabilizing RNA polymerase binding
[The typical prokaryotic promoter]
Typical distances between the start point ~10 and ~35 box
Start point of Pribnow box is actually 5-9 bp
Distance between TATA-box and ~35 box is between 16-19 bp
What is the overal process of Prokaryote Transcription?
- Polymerase bound in non-specific manner, scanning DNA
- Recognition of ~10 and ~35 sequences
- DNA in unwound around the initiation site (DNA melting Open promoter complex)
- RNA polymerase will resume movement downstream and start addding nucleotides (promoter clearance)
Explain termination for rho dependent or rho independent
- After addition of 8-9 nucleotides, the sigma SU is released and elongation phase starts
- termination can follow two different mechanisms rho-dep or rho-indep
Describe the architecture of transcribing RNA pol
- During transcription, the bubble is maintained within bacterial RNA plymerase, which unwinds and rewinds DNA
- the conditions of the partner and template DNA strand are maintained while RNA is synthesized
- B + B’ are shown to cross link with:
- template DNA
- product RNA
- ribonucleotides
- Bubble = 12-20 bp in length
- RNA-DNA hybrid region = <12 bp
- the structure was determined by protein-protein or protein-DNA crosslinking experiments
- SU B and B’ are in close proximity with the template DNA, RNA transcript and NTPs
- RNA is added at the catalytic site
- After addition and movement of the pol downstream, the DNA is rewound into a typical dsDNA
Close up on the catalytic site DNA turns at the active site
Multiple clefts, tunnels and 3-D structures maintain the DNA in single stranded conformation
The rudder in the center promotes unwinding
The bridge structure acts as a guide for the DNA towards the wall structure
The tunnel between the Bridge and Wall is the entry pont for NTPs
The NTPs are added and the RNA exits between the rudder and upper protion of the Wall
Note: the enzyme moves form left to right
What is Rho-Independent Termination?
Rho-independent is linked to actual sequence transcribed
The RNA transcript forms a stem loop structure that will promote the release of the RNA polymerase (dissociation of RNA-DNA hybrid)
GC rich area + 4 A’s
Explain Rho-dependent termination: Hexamer
and its C rich, G poor sequence without poly U, gets a small hairpin loop
Rho-dependent termination:
- The Rho factor is required. RNA polymerase transcribes DNA
- It forms an hexamer that will find a specific sequence on then transcribed RNA. Rho attaches to recognition site on RNA
- The Rho complex moves alongside following the RNA pol.
- The RNA polymerase will find a sequence referred to as terminator (C-rich and G-poor, no poly-U promotes the formation of a small hairpin loop structure that Rho complex will recognize) RNA polymerase pauses at terminator
- the Rho complex will catch up with the RNA pol.
- Rho complex will unwind the DNA-RNA hybrid in transcription bubble and the RNA will be released.
- RNA polymerase, rho and RNA are released
