Lecture 18+19 Flashcards
primary transcripts
most RNA moelcules are synthesized as biologically inactive precursors
RNA processing facts
- All tRNAS are synthesized as larger pre- tRNAs
- all large rRNAs are synthesized as a single very large precursor
- most of small non coding RNAs are modifed
- all mRNAs in eukaryotes must be modified
- most bacterial mRNAs are not modified
types of RNA processing
- cutting/cleavage, trimming, splicing
- modification
- editing
cleavage
cutting exons from introns
splicing
removing introns and gluing exons together
modification of nucleotides
5’ capping (m7 GPPP)
Polyadenylation (AAAA)
editing
base modification - change base (A –> G)
base insertion (C–>CC)
Base deletioon (GUG –> GG)
RNA processing in prokaryotes
pre-rRNA containing all rRNA sequences+ tRNAs is cleaved at the arrows by endo ribonucleases
individual pieces are trimmed by exonuclease
tRNA processing in eukaroytes
the precursors are cut and trimmed by appropriate ribonucleases
many bases in tRNAs are also modified
monocistronic (prokaryotes)
shine-dalgarno sequence in bacteria only
produces one protein
polycistronic (prokaryotes)
produces multiple proteins
All eukaryotic transcripts
synthesized as nonfunctional precursors (primary transcripts) and must be modified
RNA pol I and Pol III products
are cut and trimmed, and usually edited
RNA pol II products
ALWAYS modified
- G-cap is added at 5’ end DURING transcription
- introns are removed during and after transcription
- polyA tail is usually added at 3’ end after transcription
- some mRNAs are edited
RNA pol I product
45S RNA (pre rRNA), is processed to 5.8s, 18s, and 28s in nucleolus with an assist of snoRNAs (small nuclear RNAs)
mnay bases are modified by methylation
the transcrupt is cleaved and trimmed
tRNA procesing in eukaryotes
- both the 5’ and 3’ ends are trimmed (in all tRNAs) by RNAse P and D respectively
- CCA added to the 3’ end
- introns (when present) are removed by specialized endonulcease/ligase
- some bases are modified (in all tRNAs) to provide stability and enhance the functioning of the mature tRNA
capping
5’ ends of nascent transcripts
splicing
removal of introns
cleavage
3’ ends are generated by cleavage. NOT termination
CTD of RNAP II coordinates
- capping
- splicing
- cleavage
promoter escape
capping
elongation
splicing
eukaryotic mRNAs are capped at 5’ end
- protects 5’ end from exonuclrases
- necessary for translation
- 5’ cap: residue of 7 methylguanosine (7-MeG)
- 7-MeG is attached to the 5’ end of terminal residue of EVERY RNA via 5’-5’ - triphosphate linkage
- caps may differ by methylation pattern
1st modification state
(phosphorylation by TFIIH) of CTD allows promoter clearance and recruitment of “capping enzyme” –> Guanylyltranspherase
- the 5’ cap is formed by condensation of a molecule of GTP with triphosphate at the 5’end of the transcript (5’-5’ - Triphosphate linkage)
The guanine is subsequently methylated at N -7 and additional methyl groups are added to the 2’ hydroxyls of the first and second nucleotides adjacent to the cap
5’ cap synthesis
- Guanylyltransferase (the capping enzyme) is associated with the Pol II CTD to ensure that each mRNA is capped as it is transcribed
- Once the cap is complete, guanylyltransferase dissociates and the cap-binding complex CBC binds.
Termination of transcription RNA pol II
- Pol II termination does not occur at a conserved site or at a constant distance from the 3’ end of mature RNAs
- Mammals: take place anywhere from a few kb to several kb pairs downstream from the 3’ end of the mature transcript
- ## Polyadenylation signal (AAUAAA) is present in primary transcript. And is directly encoded by the DNAFactors responsible for cleavage: of the primary transcript bind to the AAUAAA sequence, resulting in cleavage somewhat downstream from that position.
CPSF
cleavage and polyadenylation specific factor
CstF
cleavage stimulation factor
generating 3’ end of eukaryotic mRNA
CPSF and CstF are recruited to PolyA signal sequence (AAUAA) from CTD
Endonuclease cuts just upstrewam from GU-rich sequence
addition of the 3’ poly A tail to the transcript
- the 3’ poly A tail typically 80-250 A residues
- serves as a binding site for one or more specific proteins that help protect mRNA from enzymatic destruction
- PAP (poly A polymerase) adds a stretch of A’s to the 3’ generting a poly A tail - POST TRANSCRIPTIONALLY
- Poly A binding (PABPs) bind to Poly A tail - protecting it from 3’ to 5’ exonucleases
RNA splicing
all pre mRNA splicing mechansisms consist of the ordered breaking and joining of specific phosphodiester bonds to achieve the precise excesion of introns
Splicing must be carried out quickly and correctly to produce the mRNAs required for protein production
Duchenne muscular dystrophy and cystic fibrosis
caused by aberrant pre-mRNA splicing
splice junctions
- sequences within RNA determine where splicing occurs
5’ splice site is always
GU
3’ splice site
AG
generic intron would have seuqence
GU……AG
minority of introns have AU….AC
accurate and efficient splicing relies on
base pairing between the pre-mRNA and the splicing machinery to specify the bonds to be broken or formed.
branch point (splice junctions)
approx 20-50nt upstream from 3’ splice site
the surrounding sequences and perhaps the structure of the pre-mRNA itself must play a role in the selection of splice sites.
accurate and efficient splicing relies on base pairing between pre-mRNA and the splicing machinery to specify the bonds to be broken or formed
the splice reaction
- two site speciic transesterification reactions
- resulting in phosphodiester bond cleavage and ligation - formation of lariat (lasso, loop and tail structure)
- introns released and exons are joined together
- splicing requirements: splice sites and the branch point site
these reactions are catalyzed by ribonucleoproteins (RNPs)
RNPs
complexes of non-protein coding RNAs and proteins
spliceosome - catalyzes most pre-mRNA splicing
large complex of 5 small nuclear ribonucleproteins (snRNPs snrups) + hundreds of additional protein components
-
snRNAs
single small nuclear RNAs (100-300nt) = snRNAs (U1,U2,U4-6) - in each snurp
Snurps have a name corresponding to snRNA
Each snRNA is complexed with several (under 20) proteins forming snRNPs (snurps)
the spliceosome
snRNPs (with help from proteins have several functions)
- recognition of 5’ and 3’ splice sites, and the branch site bringing those sites together; the catalysis of cleavage and joining reactions
splicing relies on many specific intermolecular reactions
protein - protein
protein - rna
rna - rna
correct splicing relies on
sequential assembly and rearrangement of spliceosome on the intron to be removed
base pairing between the snRNAs of the spliceosome and the pre-mRNA allows cells to select correct splice sites.
splicing process
- U1 binds to the 5’ splice site; U2 binds to the branch point
- U4-U5-U6 trimeric snRNP displaces U1 at the 5’ splice site, then U4 dissociates.
- U6 and U2 catalyze attack of the branch point on the 5’ splice site
- the 5’ splice site attacks the 3’ splice site, completing the reaction
self splicing
- transcripts other than nuclear pre-mRNA may contain introns that will be spliced
- all non nulcear transcripts do not use spliceosome
- they are self-spliced and the catalysis is performed by the intron itself (ribozyme) No involvement of any protein enzymes
- ## these transcripts belong to two classes: group I and group II based on the mechanism of the 1st transesterification reaction
nuclear pre-mRNA
abundance: very common, used for most eukaryotic genes
mechanism: two transesterification reactions; branch site A
major and minor splicosomes
group II introns
abundance: rare; some eukaryotic genes from organelles and prokaryotes
mechanism: same as pre mRNA
RNA enzyme encoded by intron (ribozyme)
group I introns
abundance: rare; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genes
mechanism: two transesterification reactions; requires a guanine nucleoside or nucleotide cofactor (not used as a source of energy) for the firdt transesterification
group I intron mechanism
the 3-OH of guanosoine acts as a nucleophile, attacking the phosphate at the 5’ splice site
the 3-OH of the 5’ exon becomes the nucleophile, completing the reaction
group II introns mechanism
the structure of the RNA itself, rather than the assembly of multiple snRNPs, creates an active site for catalysis
splicing mechanisms and requirements
- group I nad II introns self splice
- the information for splicing including catalytic activity is present in group I and II introns
- all 3 classes of splicing reactions proceed by two transesterification
- most of the self splicing intron sequence is critical
