post transcriptional modifications
TRANSCRIPT
POST TRANSCRIPTIONAL MODIFICATIONS
Prepared by: Narasimha Reddy.P.K
(2014-11-104)college of horticulture
kerala agricultural universityVellanikkara,thrissur
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Eukaryotic vs. Prokaryotic Transcription
• In eukaryotes, transcription and translation occur in separate
compartments.
• In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is
usually monocistronic.
– Polycistronic: one mRNA codes for more than one polypeptide
– monocistronic: one mRNA codes for only one polypeptide
• “Processing” of mRNA is required in eukaryotes for the
maturation
• No processing in prokaryotes(mRNA matures on transcription)
Coupled transcription and
translationmRNA processed and transported
out of nucleus for translation
Introduction…
• Capping (addition of
a 5’ 7-methyl guanosine
cap)
•Splicing to remove
intervening sequences
(introns)
• Polyadenylation
(addition of a poly-A
tail at the 3’)
1. mRNA Processing
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Cap Functions
Cap provides:
1. Protection from some ribonucleases degradation
2. Stabilizes mRNA
3. Enhanced translation and splicing
4. Enhanced transport from nucleus to cytoplasm
• mRNA is called hnRNA (heterogenous nuclear RNA) before splicing
occurs
•The hnRNP proteins to help keep the hnRNA in a single-stranded form
and to assist in the various RNA processing reactions
• Exon and intron lengths & numbers vary in various genes
• Exon (Expressed sequences)is any segment of an interrupted gene
that is represented in the mature RNA product.
• Intron (intervening sequences )is a segment of DNA that is
transcribed, but removed from within the transcript by splicing
together the sequences (exons) on either side of it.
mRNA splicing
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Splice Junction Consensus Sequence
• GU-AG rule describes the presence of these constant dinucleotides at the first
two and last two positions of introns of nuclear genes.
• Splice sites are the sequences immediately surrounding the exon-intron boundaries
• Splicing junctions are recognized only in the correct pairwise combinations
The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.
• Splicing is mediated by a large RNPs(Ribonucleoproteins)
complex spliceosome
• Spliceosome contains a specific set of base Uracil-rich
snRNPs (small nuclear RNPs) associated with proteins
(snRNA complex with protein)
Function of snRNPs:
• Recognizing the 5’ splice site and the branch site.
• Bringing those sites together.
• Catalyzing (or helping to catalyze) the RNA cleavage.
mRNA splicing
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Spliceosome Complex• Splicing snRNPs:
• U1: 5'- site recognition
• U2: branch site recognition
• U4: forms base pairedcomplex & acts with U6
• U5: 3'- junction binding ofU4-U6 complex
• U6: complex with U4 makesspliceosome transesterase
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spliceosomes recognize introns starting with 5'-GU and ending in AG-3’
U1
3′5′
5′ splice site 3′ splice siteBranch site
AGU
Exon 1 Exon 2
U1 binds to 5′ splice site.U2 binds to branch site.
AG
3′5′
A
U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together.
U1 snRNP
U2 snRNP
3′5′
A
U5 snRNP
U4/U6 snRNP
U2
Intron loops out and exons brought
closer together
Mechanism of Spliceosome
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U1U4
3′5′
3′5′
5′ splice site is cut.
5′ end of intron is connected to the
A in the branch site to form a lariat.U1 and U4 are released.
3′ splice site is cut.
Exon 1 is connected to exon 2.
The intron (in the form of a lariat) is released along with U2, U5, and U6 (intron will be degraded).
A
A
U5U6
U5U6
U2
Intron plus U2,U5, and U6
Two connectedexonsExon 1 Exon 2
U2
Intron will be degraded and the snRNPs used
again
Mechanism of Spliceosome
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pre-mRNA are spliced in several different ways, allowing a single
gene to code for multiple proteins
The generation of different mature mRNAs from a particular type
of gene transcript can occur by varying the use of 5’- and 3’- splice
sites
Alternative splicing
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Sex determination in the Drosophila
Polyadenylation of mRNA at the 3’ end
CPSF: cleavage and polyadenylation specificity factor
binds upstream AAUAAA poly(A) Signal 5’ end.
CStF: cleavage stimulatory factor F interacts with a
downstream GU- sequence & bound with CPSF
forming a loop in RNA
CFI & CFII: cleavage factor I & II.
PAP: poly(A) polymerase stimulates cleavage at poly A
site
Bound PAP adds ≈12 A residues at a slow rate to 3’-
OH group
PABPII: poly(A)-binding protein II.
PABPII (short poly A tail) accelerates rate of addition
of A by PAP
After 200–250 A residues have been added, PABPII
signals PAP to stop polymerization
Poly (A) tail controls mRNA stability & influences
translation
• 5’ cap
• 5’ untranslated region
• Start codon
• Coding sequence
• Stop codon
• 3‘ untranslated region
• Poly A tail
Matured mRNA
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• Smallest among RNAs (75-93 nucleotides)
• Recognizes codon on mRNA
• Shows high affinity to amino acids
• Carry amino acids to the site of protein synthesis
• tRNA is transcribed by RNA polymerase III
• tRNA genes also occur in repeated copies
throughout the genome, and may contain introns.
2. tRNA
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Transfer RNA/ Soluble RNA/ supernatant RNA/ Adaptor
RNA
1. Removal of leader sequence &
trailer
2.Replacement of nucleotide
3.Modification of certain bases:
• Replacement of U residues at the
3′ end of pre-tRNA with a CCA
sequence
• Addition of methyl and
isopentenyl groups to the
heterocyclic ring of purine bases
• Methylation of the 2′-OH group
in the ribose of any residue; and
conversion of specific uridines to
dihydrouridine(D),pseudouridine(y)
4.Excision of an intron
Processing of tRNA
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Ribozyme
RNA can act as an Enzyme and catalyse reactions including its own replication
tRNA PROCESSING AND MATURATION
• In cell >80% of rRNA
• Serves to release mRNA from DNA
•Act as ribozymes in protein synthesis
• Relatively G:::C rich
• Ribosome
• Prokaryotes – 70S (50S & 30S)
• Eukaryotes – 80S (60S & 40S)
• Prokaryotes – In 50S subunits - 23S & 5S :31 proteins
In 30S subunits - 16S :21 proteins
• Eukaryotes – In 60S sub-units – 28S, 5.8S and 5S :50 proteins
In 40S sub-units – 18S :33 proteins
3. Ribosomal RNA (rRNA)
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Processing of ribosomal RNA
• Processing of 45s molecules occurs inside nucleolus
• 45s molecules tightly associated protein forming (RNPs)
• Frist cleavage: occurs at site I & remove 5’ terminal leader
sequence, produces 41s intermediate & 18s
• Second cleavage: occurs 41s intermediate at site 3’
generates 32s intermediate
• Final cleavage: separation of 32s intermediate into 28s, 5.8s
• Processed rRNA 28s, 5.8s & 18s
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Synthesis of 5S rRNA
• rDNA cistron for 5S rRNA is present outside Nucleolar
organizer
• Transcription requires RNA pol III + TFIIIA, TFIIIB &
TFIIIC