Transcript RNA polymerase I

Gene Expression in
Eukaryotes
Transcription and RNA Processing
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Three distinct RNA
polymerases
• RNA polymerase I
– larger rRNAs (28S, 18S, 5.8S)
• RNA polymerase II
– mRNAs & most small nuclear RNAs
• RNA polymerase III
– low MW RNAs (the various tRNAs & 5S rRNA)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Polymerases - complex
enzymes
• 8 - 14 distinct subunits; visible in EM
• differ in sensitivities to a-amanitin,
– highly toxic octapeptide (8 linked amino acids)
– from common poisonous mushroom Amanita
phalloides
– also the source of microfilament toxin, phalloidin
– Pol II is very sensitive, pol I not affected, pol III
medium
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Polymerases - complex
enzymes
• mushrooms poisoning
– no immediate symptomes
– liver function deteriorates over days
– no new mRNA synthesis
– may require liver transplant
• Lots of additional TF’s required
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Processing
• All RNA types (mRNA, tRNA, rRNA)
– Primary transcripts not naked RNA
– associated with proteins even as synthesized
• Requires small nuclear RNAs [snRNAs])
– >12 involved;
– 90-300 nucleotides long
– uracil-rich nucleotides
– function in nucleus
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Ribosomal RNAs
• >80% of cell RNA
• rDNA genes repeated hundreds of times
• moderately repetitive DNA
• clustered in one or a few genome
regions
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.8
Ribosomal RNAs
• the human genome has 5 rDNA clusters
– each on a different chromosome
– In interphase, the regions come together:
nucleolus
– Disappear at cell division (mitosis)
– Reappear around rDNA (nucleolar organizers)
• nucleolus mostly ribosomal subunits
– granular appearance)
– rDNA templates
– nascent rRNA transcripts)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Synthesizing rRNA precursor
• amphibian eggs large with many
nucleoli
• 2.5 mm diameter
• selectively amplify rDNA (hundreds of
nucleoli)
• needed for embryonic development
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Synthesizing rRNA precursor
• Oscar Miller, Jr., U. of Virginia, (1960s)
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gently dispersed nucleoli fibrillar cores of oocytes
large circular fiber
resembled chain of Christmas trees
several distinct rDNA genes
arranged 1 after other (tandem repeat)
Each fiber in Christmas tree is nascent rRNA
RNA fibrils contain clumps & associated particles
• convert precursors into final rRNA products
• assemble them into ribosomal subunits
– Nontranscribed spacers between rDNA
– Also spacers between tRNA & histone
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.12
Processing of rRNA precursor –
4 rRNAs
• 3 rRNAs in large subunit (28S, 5.8S, 5S)
• 1 in small (18S)
• S value (Svedberg unit)
• sedimentation coefficient of RNA
– 28S, 18S, 5.8S & 5S RNAs are
– 5,000, 2,000, 160 & 120 bases long respectively
• 28S, 5.8S & 5S from same human pre-rRNA
– by nucleases at specific sites
– 5S rRNA from separate precursor outside nucleolus
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Processing of rRNA precursor
•
•
•
•
Pre-rRNA has lots of modified nucleotides
methylated nucleotides (>100)
pseudo-uridines (~95)
done posttranscriptionally
– conserved during vertebrate evolution
– only unaltered parts discarded during processing
– CH3 groups may
• protect parts of pre-RNA from cleavage
• promote folding into final 3-D structure
• promote rRNA interactions with other molecules
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Processing of rRNA precursor
• Pulse-chase with 14C-methionine
– 45S peaks in nucleolar material after 10 min
– 32S peaks in nucleolar material after 40-150 min
– 32S converted to 28S
– other product, 18S rRNA, in cytoplasm within 40 min
– After 2 or more hours, nearly all of radioactivity has
left nucleolus & most has accumulated in cytoplasmic
28S & 18S rRNAs
– Radiolabel in in 4S RNA peak of cytoplasm
represents CH3 groups transferred to small tRNAs
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.13
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.14
small, nucleolar RNAs (snoRNAs)
• packaged with proteins: snoRNPs
• snoRNPs associate with nascent rRNA
precursor
• first to attach contains U3 snoRNA
– binds to precursor 5' end for 5' end removal
– U3 at (~106 copies/cell) discovered long ago
– New class discovered - lower concentration
(~104 copies/cell)
– relatively long (10-21 nucleotides)
complementary
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
small, nucleolar RNAs
(snoRNAs)
• Other antisense snoRNAs
– encoded within intervening sequences of other genes
– binds to specific portion of pre-rRNA
– required to modify a particular nucleotides
– 200 different antisense snoRNAs
– one for each methylated or pseudouridylated site
– Box C/D snoRNAs - methylation
– Box H/ACA snoRNAs - pseudouridines
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.15
Ribosomal subunit assembly
• Done in nucleolus
• 2 protein types associate with rRNA as it's
processed
– Proteins that remain in ribosomal subunits
– proteins that have transient interaction with
rRNA
• needed for processing
• proteins that protect sites from cleavage
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
5S rRNA synthesis & processing
(~120 bases long)
• part of prokaryote & eukaryote large ribosomal
subunit
• In eukaryotes
– 5S rRNA is encoded by large number of identical genes
– separate from the other rRNA genes
– located outside the nucleolus
– organized in tandem array with spacers
– Transcribed by RNA polymerase III
– 5' end of 1° transcript is identical to mature 5S rRNA
– 3' end removed during processing
– 5S rRNA is transported to nucleolus
– participates in ribosome subunit assembly
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
5S rRNA synthesis & processing
• Polymerase III action
– binds to promoter within gene rather than
upstream
– Remove 5' flanking region —> still transcribed
– Delete central part (~50-80 bp) —> no
transcription
– internal promoter works elsewhere in genome
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Transfer RNAs
• ~50 tRNAs in plant & animal cells,
• each encoded by repeated DNA
sequences
• yeast: ~275, fruit flies: ~850, humans:
~1,300
– small clusters, dispersed
– contain multiple copies of different tRNA genes
– nontranscribed spacers separate tRNA genes
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.16
Transfer RNAs
• Transcribed by polymerase III
– 1° transcript of tRNA is bigger than final product
– both 5' & 3' trimming (& sometimes an interior piece)
– Ribonuclease P - found in both bacterial & eukaryotic
cells
– consists of RNA & protein subunits
• All tRNAs have triplet CCA sequence at 3' end
– added enzymatically after processing
– plays key role in protein synthesis
– charged at 3’end
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E