2.2 Expression of Genetic Information Flashcards
Role of snoRNAs
- Small nucleolar RNAs (snoRNAs) complex with protein to form snoRNPs
- U3 snoRNA binds to 5’ terminus
- Catalyzes removal of 5’ end of transcript
2 classes of snoRNA
box c/d is for methylation, (guide sequence, guides the sequence responcible for methylation)
-Antisense because they’re complementary to the conserved regions that they’re targeting
Box H/ACA snoRNAs – conversion of uridine to pseudouridine
-Partially complimentary, the area that’s not forms the loop structure (the loop is a guide that converts the uradine opposite to sudo-uranaine)
5S rRNA Processing
- Genes are separate from other rRNA genes
- Located outside nucleolus
- Organized in tandem arrays
- Transcribed by RNA Pol III
- Promoter is internal
- 5S rRNA transported back to nucleolus following processing
tRNA Processing
- ~50 different species of tRNAs
- Found in clusters throughout genome
- Transcribed by RNA Pol III
- Internal promoter
- Primary transcript is processed following trasciption (RNase P)
- primary transcript of tRNA is bigger than the final product and pieces on the 5’ and 3’ end must be trimmed away
- RNAseP catalytic component is found within the RNA component
mRNA Transcription
- mRNA transcripts transcribed by RNA Pol II
- Initiation depends on transcription factors
- Promoters are upstream of coding sequence
- Core promoter element between 24 and 32 bases upstream
- TATA Box
- Site of formation of initiation complex
- Tata Bidning Protein inserts into a groove of the double hell and bends it more that 80º
Formation of Preinitiation Complex
-AFIIA/D/B provide a platform for RNA Pol II to bind on with TFIIF
-trasciption factors recponcible for bending the DNA bend it 80º called architectural factors once that’s in place then TFII E H join and it transforms into an active trascibing machine
once trasciption begins some of the GTFs may be left behind at the promoter or released. As long as TFIID is bound to promoter, more RNA pol can attach for more transcription
-TFIIB provides binding site for RNA Pol II
TFIIF contains subunit homolgous to bacterial sigma factor
TFIIH is a multisubunit protein with enzymatic properties (kinase)
Phosphorylation of RNA Pol II
-CTD = C terminal domain of RNA Pol II
-tail is full of serines residues which are targets of kinase residues.
-CTD has 7 a.a that just repeats over
all added phosphate groups for RNA pol are on DTD which is heavily phosphorylated
TFIIH phosphoraltes series resides at #5 (this may act a a way for that uncouples the enzyme from the GTF allowing ir ro escape the preinitionation complex and move along the game being transcribed)
anotherkinease phophoralates the series on #2 (facilitate recruitment of additional portion factors
CTD is platform for dynamic loss and gain of factors of formation of a mature mRNA
mRNA Structure
- Encode a specific polypeptide
- Found in cytoplasm
- Attached to ribosomes
- Contain noncoding segments (untranslated)
- Eukaryotic mRNAs are modified t 3’ and 5’ ends
- 5’ methyl guanosine cap
- 3’ poly A tail
Discovery of “Split Genes”
- hnRNAs precurcer to mRNA
- Adenovirus: the leader sequence is transcribed from 3 distance speterate segments of DNA (not complementary to a repeated sequence) (between the blocks in interviewing sequences are not in the mRNA
- the presents of genes with interlining sequences called split genes is the rule not the exception
- the part of a split gene that contribute to mature RNA is the exon and the intervening sequence is the intron
- widespread in eukaryotes
- …so how does mRNA precise sequences without introns?
- idea 1: cells produce a primary trascipt of the entire trasciption unit and the portions of introns are then removed
- this would explain why hnRNA is so large
Describe the steps by which eukaryotic mRNA is modified at the 5’ and the 3’ ends (one is 3 steps one is two steps)
Capping (Capping Enzyme)
1) 5’ end of nascent pre-mRNA binds to a capping enzyme removes phosphate RNA Triphosphatase)
2) adds guanine residue Guanylyltransferse
3) Methytransferases add methyl group to terminal guanosine cap and the ribose sugar RNA Methyltransferase
Polyadenylation
A) Endonuclease cleaves RNA strand generating new 3’ end
B) Poly A polymerase adds adenosine residues to the 3’ end (non-template driven)
Conserved Regions of RNA
- AGGU these are conserved sequences at the boundaries you typically find these sequences
- at 3’ end you have polypyrididine tract C’s and U’s (good give away of an intron)
- you want to devise gene geneqeunces that can predict the area of introns.
- First step, 2’Adenine has a 2’ hydrozul group that can make a attack on the 5’ spice site (is in proximity due to secondary structure) makes nucleophilic attack and links to the guanine 2’ OH
Group II Intron Splicing Mechanism
First step, 2’Adenine has a 2’ hydrozul group that can make a attack on the 5’ spice site (is in proximity due to secondary structure) makes nucleophilic attack and links to the guanine 2’ OH
liberates the 3’ OH on the 5’ that can make an attack on the axon and covalently link them tougher and the lariat is releases (the intron released as a lariat)
Self-Splicing Introns
Thomas Cech
determine the machinery responsible for splicing. Must be protein since proteins do most things. Had a terahymen model.
Did cell free assay, extra and mix with RNA and say if they could get splicing to work…it did.
So what are the proteins in the solution responsible for splicing? did it very one by one.
Had a negative control, (splicing won’t work since you removed all the protein..just RNA temlate…shouldn’t spice)…while this spiced quite well.
Anomie sulfate participates proteins…starts spicing better one you take the proteins out.
okay lets do it again…but we’ll heat denature it..what the heck it works
therefore splicing must be done by the RNA
Spliceosomal Intron Mechanism
- U1 snRNP attaches at 5’ branch site
- U2 recruited by U2AF
- binds to poly-pyrimidine tract
- U2 snRNP attaches at Adenine branch site
- Forces the adening to bulge outward which makes it more likley to do the nucleophilic attack
- ESE is exon splicing enhancer (sequence within the exon)
- Binding of U4/U6 and U5 snRNPs
- U6 has catalytic (ribozyme) activity
- U6 activity is inhibited as long as U4 is base pared to it
- beginning of the formation of the lariat
- Displacement of U1 by U6
- Dissociation of U4 from U6
- U6 + U5 have catalytic activity (helices)
- pull close to each other, so you get nucleophilic attack
- Cleavage of 5’ splice site and formation of lariat intron-3’ exon intermediate
- Exon 1 and 2 kept together by U5 SNRP
- now 2nd rx where exon 1 nucleophilic attacks on exon 2 with aid of U5 SRP and the remnant lariat
- Second cleavage reaction at 3’ splice site and simultaneous joining of exons.
Spliceosomal vs. Self-Splicing Introns
shows how spiceosome looks similar to self spicing
- self spicing is one conitinuould RNA sequence
- spliceosome is a combo of ssnRPS and the actual intron
- Evidence that spliceosome derives from self splicing
