Transcript 5` splice site

Gene Expression in
Eukaryotes
mRNA Transcription and Processing
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Eukaryotic Transcription
Machinery
• RNA polymerase II
– synthesizes all eukaryotic mRNA precursors
– composed of 12 different subunits
– remarkably conserved from yeast to mammals
• Polymerase II promoters
– 5' side of each transcription unit (mostly)
– TATA box at 24-32 bases upstream from start site
– Consensus: 5'-TATAAA-3'
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Figure 11.18a
Eukaryotic Transcription
Machinery
• Initiation of transcription
– Requires a number of general transcription factors (GTFs)
– Their precise roles remain to be determined
– General = conserved in a variety of genes and organisms
– A preinitiation complex assembles at the TATA box
• Required before Pol II binds
– First: TATA-binding protein (TBP)
• TBP has 10-stranded b sheet curved into a
• saddle-shaped structure that sits astride the DNA
• TBP is subunit of TFIID (fraction D)
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Eukaryotic Transcription
Machinery
• TBP binding distorts DNA conformation
– Bound DNA develops a distinct kink
– DNA duplex becomes unwound over ~8 bp
• TBP is a universal TF
– mediates binding of all 3 eukaryotic RNA polymerases;
– present in 3 different protein complexes
• TFIID (pol II)
• SL1 (pol I)
• TFIIIB (pol III)
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GTF’s and initiation
• 3 GTFs interact with promoter on DNA
– TBP of TFIID
– TFIIA
– TFIIB)
• provide platform for multisubunit
polymerase + TFIIF
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GTF’s and initiation
• followed by another pair of GTFs
– TFIIE & TFIIH
– TFIIH is only GTF known to possess enzyme
activities
• 2 of its subunits act as DNA helicases
• allows polymerase access to template strand
– Another TFIIH subunit functions as protein
kinase
• phosphorylate RNA polymerase
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Figure 11.19
Elongation
– One GTF (TFIID) may be left behind at
promoter
• Future initiation?
– Other GTF’s are released from the complex
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(CTD) of largest polymerase II subunit
•
7 amino acid sequence (-Tyr-Ser-Pro-Thr-Ser-Pro-Ser-);
•
in mammals, 52 repeats
•
All but 2 prolines are targets for phosphorylation
•
preinitiation pol II is nonphosphorylated
•
when transcribing, it is heavily phosphorylated
•
phosphorylation is likely a trigger for transcription
•
An elongation complex
– a number of large accessory proteins
– > 50 components
– total molecular mass of >3 million daltons
•
Probably, template moves through immobilized machine
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Figure 11.20
Regulation
• Specific transcription factors
– bind to other sequences (CAAT-box, GC-box,
enhancers)
– activate (or prevent) preinitiation complex
formation
– Determine polymerase initiation rate
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mRNA properties
•
They are found in the cytoplasm
•
They are attached to ribosomes when they are translated
•
significant noncoding, nontranslated segments
– ~25% of each globin mRNA is noncoding
– Noncoding portions are found on both 5' & 3' ends
– have sequences with important regulatory roles
•
altered ends not seen in prokaryotes
– 5' end - methylated guanosine cap
– 3' end - string of 50 - 250 adenosine residues [the poly(A) tail]
– histones are an exception
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Figure 11.21
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Figure 11.22c
Split Genes
• Philip Sharp et. al. (MIT)
• Richard Roberts, Louise Chow et. al. (Cold Spring
Harbor, NY)
– Adenovirus introns
• Alec Jeffreys & Richard Flavell (1977, U. of
Amsterdam)
– Exons - parts of gene that contribute to mature RNA
product
– b-globin gene
– Introns - intervening sequences
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Figure 11.23
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Figure 11.24
Split Genes
• Split genes found in simpler eukaryotes (yeast,
protists)
– fewer in number & smaller in size than in plants & animals
– Introns are found in all types of genes (tRNAs, rRNAs,
mRNAs)
• Must be removed from primary transcript to make
mature mRNA
• Shirley Tilghman, Philip Leder, et al. (NIH) –
– R-loop formation seen in EM
– determined relationship between 15S & 10S (mature) globin
RNAs
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Figure 11.25a & b
Split Genes
– Ovalbumin (protein found in hen's eggs)
gene
• 7 loops form; correspond to 7 introns
• ~3 times more sequence than 8 exons
• Individual exons in all genes are typically < 300 bases
• Individual introns typically between 1,000 & 100,000
bases
• Explains hnRNA length
– Type I collagen gene
• >20 times the length of mature message
• contains >50 Karp/CELL
introns
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Processing
•
Ribonucleoproteins convert transcript to mature mRNA
•
Addition of a 5' cap & a 3' poly(A) tail
•
Removal of any intervening introns
•
5' methylguanosine cap forms very soon after RNA
synthesis begins
– 5' end initially has triphosphate
– First enzyme produces diphosphate
– Then, GMP is added in inverted orientation
– 5'-5' triphosphate bridge
– Next, guanosine base is methylated at position 7
– ribose methylated at 2' position
– 5' end modifications occur very quickly
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Processing
– May serve several functions:
• prevents exonuclease digestion of mRNA 5' end
• aids in transport of mRNA out of nucleus
• important in initiation of mRNA translation
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The poly(A) tail
– ~15 bases downstream from AAUAAA
– a protein complex carries out processing at 3' end
• associated with the RNA polymerase
• Included is an endonuclease
• poly(A) polymerase adds ~250 adenosines
– protects the mRNA from premature degradation
– Poly(A) tail allows affinity chromatography
purification
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Figure 11.28
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Figure 11.29
Splicing
– Must be absolutely precise
– single base error changes reading frame
– conserved sequence found at exon-intron
junctions
– Usually G/GU at 5' intron end (5' splice site)
– Usually AG/G at 3' end (3' splice site)
– ~1% of introns have AT & AC, respectively
– processed by different spliceosome (U12
spliceosome)
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Figure 11.30
Splicing
• functional differences as yet undetected)
– has U12 snRNA instead of the U2 snRNA of the major
spliceosome
– U12 spliceosomes
• absent from yeast & nematodes
• present in plants, insects & vertebrates
• regions adjacent to intron contain preferred
nucleotides
• play big role in splice site recognition (exonic
enhancers)
• mutation can block intron excision
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Splicing
• human thalassemia caused by
mutations in globin splice sites
• RNA catalytic abilities led to
understanding of splicing mechanism
• Thomas Cech et al. (1982, U. of
Colorado)
– RNA catalysis in pre-rRNA
– ciliated protozoan Tetrahymena (ribozymes)
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Two types of intron splicing
mechanisms described
• Group I introns (Tetrahymena pre-rRNA)
– most common in fungal/plant mitochondria, plant
chloroplasts, & in nuclear RNA of lower
eukaryotes, like Tetrahymena
– variable sequence, but similar 3D structures
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Two types of splicing
– Group II introns
• also self-splicing
• seen in fungal mitochondria & plant chloroplasts
• structure very complex & different from Group I introns
• go through intermediate stage (lariat, like cowboy rope)
– First step is cleavage of 5' splice site
– followed by formation of lariat
– covalent bond between intron 5' end & A near 3' end
– 3' splice site cleavage releases lariat
– allows exon cut ends to be ligated
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Figure 11.32
Two types of splicing
– Animal pre-mRNAs are processed like Group
II introns
• difference is that intron cannot splice itself
• needs snRNAs & their associated proteins
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hnRNA’s
• hnRNP’s facilitate processing reactions
– spliceosomes remove introns
• have a variety of proteins & snRNPs
• assembled they bind to the pre-mRNA
– snRNPs help remove introns from transcript
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hnRNP’s
• snRNPs required:
– U1 snRNP, U2 snRNP, U5 snRNP & U4/U6
snRNP (U4 & U6 snRNAs bound together)
– U6 is most likely to act as a ribozyme
– makes both cuts in the pre-mRNA required for
intron removal
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Figure 11.35
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Figure 11.36
snRNPs: a dozen or more
proteins
• One family, the Sm proteins are present
in all of the snRNPs
• they bind to one another & to a
conserved site on each snRNA
• forms the core of the snRNP
• Sm are targets of autoimmune
antibodies
– systemic lupus erythematosus
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snRNP’s
• The other proteins of the snRNPs are unique to each
particle
– ATP-consuming, RNA helicases (unwind double-stranded
RNAs)
– helicases are found within snRNPs
– at least 8 implicated in the splicing of yeast pre-mRNAs
• snRNAs are catalytically active (not the proteins)
– similar to group II introns, which splice themselves
– snRNAs closely resemble parts of the group II introns
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snRNP’s
• The proteins likely serve supplementary
roles
– Maintaining the proper 3D structure of the
snRNA
– Driving changes in snRNA conformation
– Transporting spliced mRNAs to the nuclear
envelope
– Selecting splice sites to be used
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snRNP’s
• snRNP proteins - not alone in mRNA processing
– SR proteins: large number of SR dipeptides
– thought to form interlacing networks that span
intron/exon borders
• They help recruit snRNPs to the splice sites
• SR proteins have positive charge
• may also bind electrostatically to negatively charged
phosphate
• assembly of splicing machinery occurs during RNA synthesis
• most of RNA processing machinery travels with polymerase
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Figure 11.37
snRNP’s
– Most genes contain a number of introns
– splicing reactions occur repeatedly on single
1° transcript
• introns may be removed in preferred order
• generates specific processing intermediates
whose size lies between
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