Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 distinct subunits; visible in EM differ in sensitivities to  -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; – 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 Figure 11.8

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 11.12

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 –CH 3 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 14 C-methionine –45S peaks in nucleolar material after 10 min –32S peaks in nucleolar material after 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 CH 3 groups transferred to small tRNAs

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

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 (~10 6 copies/cell) discovered long ago –New class discovered - lower concentration (~10 4 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 Figure 11.15

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 11.16

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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