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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) – – – – – – – 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