transcription
Transcription
The importance of transcription processes is reflected as: a Molecular machine, highly regulated, and couple to other cellular processes.
RNA can be as diverse as proteins in terms of functionality!
Pol II is the enzyme responsible for transcribing genes that encode proteins in eukaryotes.
Cis and trans elements in transcription:
Cis element are located on the genome and are either proximal (core promoters) or distal
(enhancer). The cis elements are genetic sites that trans-regulatory elements can bind to exert their
functions.
Trans elements are proteins that bind cis regulatory elements and affects transcription.
RNA transcription:
Preinitiation complex (PIC): basal transcription factors form complex on the core
promoter leading to recruiting RNA Pol II.
Basal transcription factors: TFIID (Transcription factor), TFIIB, TFIIA, TFIIH, TFIIE
TFIID: is a complex of nine proteins in yeast, one of them is TBP (TATA binding protein) that Bind TATA cis element, causes an almost 90 degree bend in DNA.
TFIIB: is the second general transcription factor that enters the PIC. It creates a
TFIID/TFIIB interaction platform through TBP. It stabilize TBT-TATA binding,
effects start site selection, has zinc ribbon
TFIIF: brings Pol II to the complex, it recognizes TFIID/TFIIB platform, it is a
heterotetramer complex. It also shown to stabilize the PIC and destablise nonspecific
Interaction
TFII E: Recruits TFII H, function in promoter melting and clearance, zinc binding domain
TFIIH: It is the helicase, it also could phosphorylate POl II, DNA dependent ATPases,
TFIIA: shown to stimulate transcription by stabilizing TBP onto TATA target,
Transcription initiation as whole:
There have been several models for describing the transcription in eukaryotes.
There have been also evidences for supporting each model (see slides)
a) step wise assembly:
b) preassembled initiation complex
c) Nuclear compartmentalization assisted
Wrapping DNA by initiation complex:
It has been reasons and experimentally shown that initiation complex wraps DNA, helping to create the open Complex and allowing distance region to come together. Both TFIIH and TFII E play role in wrapping the DNA.
RNA Pol II:
The enzyme that does catalyze the mRNA syntheses. In eukaryotes, it is made of 12 subunits (see slide).
RNA pol II structure: 10 subunit structure of POl II was done without the RB7 and RB9
left: the cavity that the enzyme forms, 60 Ang., about 17-19 base pair of B DNA
Clamp: is important part of polymerase, change conformation from open to close, in close conformation RNA/DNA are unable to escape, there are 5 switches on clamp that change conformation upon closing clamp, clamp is both flexible and stable, it also interacts with exiting RNA and the bridging helix (see slide and the paper)
Jaws: are mobile, allow some freedom of movement of DNA downstream.
Bridging helix: links the borders of cleft, change conformation (especially in bacteria), acts to separate active base from next downstream base. Active base (is on DNA template that H. bonds to incoming ribonucleotides, is flipped by 90 degree)
Wall: prevent DNA from entering and existing as liner, is part of Rpb2
Pore: lies below the active site, where nucleotides and substrate enter the molecule
RNA exist channel: formed by one side of clamp and another domain called Flap, is able to open and close
CTD (carboxy terminal domain): it is made of heptide repeats (Y1S2P3T4S5P6S7) more than 50 in human, it interacts with Mediators, elongation factor, capping enzyme, termination factor and more. The heptopeotide that has three Serine residues gets phosphorylated on the 3 residue
Existence of three modules in RNA poI:
1) Jaw-lob module: DNA first enters the Pol II through this chamber., can takes up 15-20 bp of downstream DNA (viewed from the POL II enzyme)., this is without melting DNA.
2) Cleft chamber: the DNA melts as enter the second chamber, brings DNA close to the active site
3) RNA exit channel: wall of proteins of pol II blocks extension of RNA-DNA hybrid, DNA is pushed into the third chamber., RNA (the product) is pushed into this camber, DNA template goes through different opening which will be annealed with the non-template strand
Mediators:
-Mediators are proteins that have found to interact with Pol II enzyme.
-They were first identified as suppressor mutant of pol II CTD; that is they were found to interact with CTD domain.
-They form huge complexes that mediate the interaction of transcriptional activators and POl II
- They are capable of forming different complexes as to activate or repress transcription
POL II holoenzymes:
-a complex(s) that would mimic the in vivo transcription activator effect in vitro
-is pol II + GTF + mediator, is a huge complex acting at the transcription.
- In mammalian other factors such as splicing factors have been found in the holoenzyme complex!
Elongation:
Elongation is when the Pol II is able to escape promoter, and elongate the transcript.
Elongation factors: are factors that can affect elongation in a positive or negative manner.
Examples of elongation factors: TFIIS, P-TEFb, elongator, DSIF, Fcp1, Elongin..
How could elongation factor effect elongation? (You may think of how it can influence transcription)
One way is to affect the phosphorylation state of CTD like p-TEF-b (positive transcription elongation factor) that joins up with a kinase that affects CTD phosphorylation.
-TFllS- another elongation factor: is stimulatory factor. When transcription is blocked, it stimulate RNA pl II to cleave RNA and to restart transcription.
Transcriptional pausing and promoter escape:
-RNA Pols usually have pauses and also generate abortive transcripts
- 3-4 or 8-9 nucleotide transcripts are made: abortive transcripts
- after 10 and 15-16 nucleotide the RNA-DNA hybrid is stabilized: promoter clearance
- after this stage the elongation cycle can take a full swing
- the clearance can provide regulation to the elongation step
- some cis elements upstream sequences could be important for clearing the promoter (see slide)
Transcription activators:
- Are factors that bind cis element named enhancer (or upstream activating sequence in yeast (UAS) ),
- Are tissue or conditions specific
- They could interact with co-regulators like those modifying chromatin enzymes
- They could bring about looping of chromosome
-They may contain various domains to bring about their action: (see text book for more details:
Zinc finger (Cys2-His2 zinc finger) zinc ion is tetrahydrally liganded by His and Cys
Leucine zipper: contain Leu at every 7th position. These Lue bring dimerization domain
Helix-loop-helix (HLH): that bind major groove of DNA.
Regulation of transcription:
Due to its importance, the transcription can be regulated in many steps. That is at the initiation, elongation, and termination/processing. However, one of the major ways to regulate transcription is at through epigenetic control.
Epigenetic control of transcription:
-Epigenetic are those changes that happen independent of DNA sequences;
-they are two kinds of epigenetic changes: one is the modification that happen on histone/nucleosome. Second those modifications that take place on DNA namely: methylation.
Chromatin role in gene expression:
- In general chromatin have negative role in gene expression., in order for a gene expression to happen in eukaryotes, the chromatin need to be opened up and altered. This happens through modifying enzyme such as histone deacylatases and ATP dependent nucleosome sliding complexes.
DNA methylation:
-DNA can be methylated on GC island (see slide)
- It has been found that the methylation can mainly repress methylation
- it is shown that methylation of DNA can block RNA Pol to move on the DNA
RNA processing:
RNA gets processed at its 5’ and ’ end. Both of these processes are important for RNA biogenesis and cell survival.
5’ capping:
The 5’ of RNA get capped in three enzymatic steps (see slide)
- Hydrolyzing the triphosphate end (performed by RNA triphosphatase)
- capping diphosphate (carried out by Guanylyltransferase)
- Methylating the N7 of Guanine (executed by RNA methyltransferase)
Why cap the message? To stabilize it, role in splicing, transport and translation
The capping happens co-transcriptionally (see slide):
- Capping happen in the early stages of transcription
- RNA guanyltransferase bind phspho-Ser5 in CTD of PO lI, recruits triphosphotase
- the methyltransferase bind phosphor-Ser2 that is phosphorylated late during transcription
3’ polyadenylation and termination of the message:
The 3’ end message need to be terminated and also gets poly A attachments. These two processes are somehow connected (coupled):
- they share some components,
-both happen at 3’ end
Two models (coupling termination and 3’ polyadenylation):
- Antiterminator model: polyaddenylation brings about dissociation of elongation
factors and recruiting termination factor
-Torpedo model: polyadynlation machinary cleave the RNA and generate unportectd 5’ end RNA, which is then recognized by RNA nucleases which is then destroy RNA and push pol II out of DNA, leading in termination of transcription.
Polyadenylation machinery:
- Cis factor involve (see slides for detail)
-Trans factor involve: leading to the pol I polymerase to add up to 200 adenines
CTD of RNA pol II is involved in cleavage/polyadenylation (see slide)
RNA splicing
-Eukaryotic mRNA was soon determined to be different with the primary nuclear RNA.
-The mRNA is shorter and contain less sequences than its original transcribed (spliced)
-Splicing is important process that is highly regulated, and it is coupled to the transcription.
Spliceosome:
-a large complex, made of proteins and 5 small neclearRNA (sno)
-it carries out two transesterifications (see slide)
-there are some cis regulatory element that splieosome acts on (see slide)
- there exist different complexes that through which spliceosome executes its function (see slide)
- Coordination and structural rearrangement (RNA and protein) are highligheted in its work
- ATP is required for the work of splicesome
- are certain domains that are crucial in its function: (For example: DEAD, RS)
-There are mechanical devices that execute spliceosome in its function
Alternative splicing:
-There are several forms of alternative splicing (see slide)
- There are some cis elements involve in alternating splicing
- Why to make alternative splicing?
RNA export
After mRNA is transcribed, processed at both ends, and spliced, it gets transported out of the nucleus to the cytoplasm for translation.
-There exist pathway that communicate in concert between cytoplasm and nucleus
- there could be control in transporting RNA to the cytoplasm or vice-versa
- It has turned out that foe each RNA there exist different pathway (see slide)
Nucler porin complex:
-One of the first obstacles for RNA (or any macromolecule) to exit nucleus is the nuclear membrane.
-There are pores in the nuclear membrane that through those molecules could go out
- These pores form basket like shape that are made of specific proteins (see slides for detail)
Karyopherin-RanGTpase:
- RNAs other than mRNA are transported by proteins name karyopherins
- These proteins use cofactor named Ran proteins for loading and unloading
- Ran proteins are small GTPase proteins that hydrolyze GTP
- There are protein called GTPase activating protein (GTP) that help Ran to hydrolyze GTP at the greater spped
- There are also proteins called Guanine exchange factor (GEF) that enable Ran to exchange GDP with GTP.
mRNA export:
mRNA uses different route for export; it is Ran independent. Its essential component are:
-Proteins that recognize mRNA (Yar1)
-export receptors (Mex67p/Tap heterodimer)
- a motor: to push RNA out of nuclear porine complex (Dbp5)
There are some specific domains involve in mRNA export (for detail see slides)
Protein translation
-Protein translation is where mRNA, tRNA, rRNA, and proteins meet.
-Ribosome is the site of translation
-Ribosome could be the most efficient macromolecular machine!
-Ribosome carries out the peptidyl transfer reaction
Structure of Ribosome:
-It is both flexible and dynamic
-it is made of protein (1/3) and RNA (2/3)
-the structure is mainly determined by RNA (helices and RNA-RNA interaction)
-There are protuberances in the structure (see slide)
-There can locate three sites: A, P, E that are important in translation
-There are special folds that are present in ribosome (see slide)
-RNA-Protein interaction is mainly electrostatic (see slide)
Stages of translations:
-initiation, elongation, termination, recycling (see slides for details)
-Translational factors (EF-Tu, EF-G) do carry out important functions
Macro Molecular mimicry:
-When protein mimic nucleic acid shape
-is a mechanism that overly used in protein translation (see slides)
- mimicking the shape dictates the function
How is accuracy maintained in translation?
-selection of correct amino acids (codon-anticodon interaction)
-accurate translocation of tRNA and mRNA
The monitoring of accuracy happens via two steps: initial selection and proofreading
-Initial selection: ribosome senses the correct base pair from cognate and near cognate,
This requires the ribosome make special contacts with the minor groove of the first and second bases of anticodon., mismatch introduces different geometry (shape) that have different intereaction (h-bonding) with the decoding center in ribosome
-Proofreading: cognate aa-tRNA to stimulate GTPase activity of EF-Tu much faster than noncognate, near cognate aa-tRNA that bind mRNA cannot hydrolyse GTP as well as cognate. The hydrolysis brings conformational changes that bring about correct codon-anticodon selection. Ribosomal proteins (L7/L12) that stimulate GTPase hydrolysis are also important in proofreading.
Role of 30 S (the decoding center):
-local conformation changes activation, help better placing the correct codon-anticodon in the decoding center (which is located on 30S subunit). The local conformational changes ultimately lead global conformational changes on 30s ribosome:(like rotation of its shoulder, towards interspace subunit) and closing onto the cognate a.a tRNA. Conformational Changes in 30s subunit can be transferred to 50s subunit (the GTPase activation center).
The ribosome uses induced-fit mechanism for specificity. Changes of ribosome do not take place with incorrect substrate and are structurally different with correct substrate. Binding of correct substrate induced changes that orient and active site for proper reaction which these changes are not seen with the incorrect substrate.
