Transcription Flashcards
(27 cards)
Transcription
Copy of coding DNA into a single stranded RNA. Requires an Rna polymerase, dsDNA template, nucleotides. polymerase catalyses the formation of the phosphodiester bonds. No primer and RNA grows 5’ to 3’. RNA is direct copy of sense strand
Prokaryotic transcription RNA polymerase
Two large subunits beta and beta’ (nucleotide binding and template binding respectively) and two smaller subunits (both alpha involved in enzyme assembly) and a sigma factor.
Sigma factor
Sigma factors bind RNA polymerase and promoter sequences bringing them into contact to form initiation complex (covering 75-80bp either side of the start point). Different sigma factors bind to different promotors. E.g. rpoD gene produces sigma factor 70. this enables RNA polymerase to bind DNA at -50 to +20 (TTGACA -35 sequence, TATAAT -10 sequence). Acts as initiation factor staying at beginning of transcription
Initial elongation complex
loses sigma, forms at ten bases and loses contact with the -35 to -55 region
General elongation complex
forms at 15-20 bases and covers 30-40 bps
Stages of transcription
template recognition by polymerase, unwound at promotor, initiation, elongation, termination
Intrinsic termination
Intrinsic termination: The terminator sequence is usually a pallindromic sequencethat forms a stem-loophairpinstructure that leads to the dissociation of the RNApolymerase from the DNA template.
Rho dependent termination
atermination factorcalledρ factor (rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNApolymerase. A stem loop structure upstream of the terminator region pauses the RNAP, when ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription
Classes of RNA
mRNA information, rRNA structural, tRNA informational/structural, RNP functional ribonucleoprotein. All transcribed from DNA and form receive post transcriptional changes
prokaryotic mRNA
T1/2 for prokaryotic mRNAs a few mins. Mostly polycistronic
RNA processing
bases can be modified, All stable RNAs are processed (mRNA rarely) Approx 10 nucleases involved Enzymes mainly recognise 3D structure Bases other than AGTC are formed. Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events. The first two are proteins, while RNase P is a ribozyme (Apirion and Miczak)
Differences in eukaryotic transcription
3 nuclear RNAPs., recognise DNA/Protein complex, 5’,3’ modified, mRNA processed from hnRNA, Monocistronic, Long T1/2
RNA polymerases
Large enzymes with two large subunits and 10 minor units
I: nulceolus, produces rRNA, 50-70% activity, not alpha aminitin sensitive
II: nucleoplasm, nuclear RNA, 20-40%, sensitive to alpha- actinin. promotors contain TATA Box and UPEs (CAAT Box, GC Box, Octamer)
III: nucleoplasm, tRNA, 10%, species specific alpha actinin
RNAPII CTD
The carboxyl end of RNA Pol II contains a stretch of seven amino acids that is RNA Pol II repeated 52 times in the mouse enzyme and 26 times in yeast. This heptapeptide has the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser and is known as the carboxy- terminal domain or CTD. These repeats are essential for viability. The CTD sequence may be phosphorylated at the serines and some tyrosines. In vitro studies have shown that the CTD is unphosphorylated at transcription initiation, but phosphorylation occurs during transcription elongation as the RNA polymerase leaves the promoter. Since RNA Pol II catalyzes the synthesis of all of the eukaryotic protein-coding genes, it is the most important RNA poly- merase for the study of differential gene expression
RNA polymerase II promotors
different combinations of TATA boxes, CAAT boxes, GC boxes and other elements. the spacing between the TATA box and the start site is important
TFIID
responsible for binding to this key promoter element. The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II tran- scription initiation complex. TFIID is a multiprotein complex in which only one polypeptide, TATA-binding protein (TBP) binds to the TATA box. The complex also contains other polypeptides known as TBP-associated factors (TAFIIs). It seems that in mammalian cells, TBP binds to the TATA box and is then joined by at least eight TAFIIs to form TFIID.
TBP
Binding of TBP deforms the DNA so that it is bent into the inside of the saddle and unwound. This results in a kink of about 45° between the first two and last two base pairs of the 8 bp TATA element.
TFIIA
TFIIA binding to TFIID prevents binding of inhibitors and allows the assembly process to continue.
TFIIB
TFIIB can also bind to the RNA polymerase. FIIB acts as a bridging factor allowing recruitment of the polymerase to the complex together with a further factor, TFIIF
TFIIF
Binds with the RNA polymerase II.
TFIIH, TFIIJ, TFIIE
These proteins are necessary for polymerase transcription in vitro and associate with the complex in a defined order. TFIIH contains both kinase and helicase activity. Activation of TFIIH results in phosphorylation of the carboxy-terminal domain (CTD) of the RNA polymerase which allows the RNA polymerase to leave the promoter region
mRNA capping eukaryotes
5-end is chemically modified by the addition of a 7-methylguanosine. addition of a GMP nucleotide to the new RNA transcript in the reverse orientation compared with the normal 3–5 linkage, giving a 5–5 triphosphate bridge. The reaction is carried out by an enzyme called mRNA guanyltransferase and there can be subsequent methylations of the sugars on the first and second transcribed nucleotides, particularly in verte- brates. The cap structure forms a barrier to 5-exonucleases and thus stabilizes the transcript
mRNA polyA eukaryotes
sequence elements of a polya signal (20bp) a cleavage and addition site, and a downstream GU rich region. specific protein factors recognize these sequence and assemble a complex, cleavage takes place and then one of the factors, poly(A) polymerase (PAP), adds up to 250 A residues to the 3-end of the cleaved pre-mRNA. The poly(A) tail on pre-mRNA is thought to help stabilize the molecule since a poly(A)-binding protein binds to it which should act to resist 3-exonuclease action. In addition, the poly(A) tail may help in the translation of the mature mRNA in the cytoplasm. Not all polyadenylated (e.g. histones)
mRNA splicing
The 5-end of almost all introns has the sequence 5-GU-3 and the 3-end. 5-AG-3. The AG at the 3-end is preceded by a pyrimidine-rich sequence called the polypyrimidine tract. About 10–40 residues upstream of the polypyrimi- dine tract is a sequence called the branchpoint sequence which is 5-CURAY-3. Different in yeast. the bond in front of the G at the 5-end of the intron at the so-called 5-splice site is attacked by the 2-hydroxyl group of the A residue of the branchpoint sequence to create a tailed circular molecule called a lariat and free exon 1. In the second step, cleavage at the 3-splice site occurs after the G of the AG, as the two exon sequences are joined together. The intron is released in the lariat form and is eventually degraded. Catalysed by U1 (5’ splice site), U2 (binds branch site) and U4, U5 and U6