Gene Expression in Eukaryotes Transcription and RNA Processing

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Gene Expression in Eukaryotes Transcription and RNA Processing Copyright, ©, 2002, John Wiley &

Gene Expression in Eukaryotes Transcription and RNA Processing Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Three distinct RNA polymerases • RNA polymerase I – larger r. RNAs (28 S,

Three distinct RNA polymerases • RNA polymerase I – larger r. RNAs (28 S, 18 S, 5. 8 S) • RNA polymerase II – m. RNAs & most small nuclear RNAs • RNA polymerase III – low MW RNAs (the various t. RNAs & 5 S r. RNA) Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Polymerases - complex enzymes • 8 - 14 distinct subunits; visible in EM •

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 3 E

Polymerases - complex enzymes • mushrooms poisoning – no immediate symptomes – liver function

Polymerases - complex enzymes • mushrooms poisoning – no immediate symptomes – liver function deteriorates over days – no new m. RNA synthesis – may require liver transplant • Lots of additional TF’s required Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Processing • All RNA types (m. RNA, t. RNA, r. RNA) – Primary transcripts

Processing • All RNA types (m. RNA, t. RNA, r. RNA) – Primary transcripts not naked RNA – associated with proteins even as synthesized • Requires small nuclear RNAs [sn. RNAs]) – >12 involved; – 90 -300 nucleotides long – uracil-rich nucleotides – function in nucleus Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Ribosomal RNAs • >80% of cell RNA • r. DNA genes repeated hundreds of

Ribosomal RNAs • >80% of cell RNA • r. DNA 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 3 E

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Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 8

Ribosomal RNAs • the human genome has 5 r. DNA clusters – each on

Ribosomal RNAs • the human genome has 5 r. DNA clusters – each on a different chromosome – In interphase, the regions come together: nucleolus – Disappear at cell division (mitosis) – Reappear around r. DNA (nucleolar organizers) • nucleolus mostly ribosomal subunits – granular appearance) – r. DNA templates – nascent r. RNA transcripts) Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Synthesizing r. RNA precursor • amphibian eggs large with many nucleoli • 2. 5

Synthesizing r. RNA precursor • amphibian eggs large with many nucleoli • 2. 5 mm diameter • selectively amplify r. DNA (hundreds of nucleoli) • needed for embryonic development Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Synthesizing r. RNA precursor • Oscar Miller, Jr. , U. of Virginia, (1960 s)

Synthesizing r. RNA precursor • Oscar Miller, Jr. , U. of Virginia, (1960 s) – – – – gently dispersed nucleoli fibrillar cores of oocytes large circular fiber resembled chain of Christmas trees several distinct r. DNA genes arranged 1 after other (tandem repeat) Each fiber in Christmas tree is nascent r. RNA fibrils contain clumps & associated particles • convert precursors into final r. RNA products • assemble them into ribosomal subunits – Nontranscribed spacers between r. DNA – Also spacers between t. RNA & histone Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 12

Processing of r. RNA precursor – 4 r. RNAs • 3 r. RNAs in

Processing of r. RNA precursor – 4 r. RNAs • 3 r. RNAs in large subunit (28 S, 5 S) • 1 in small (18 S) • S value (Svedberg unit) • sedimentation coefficient of RNA – 28 S, 18 S, 5. 8 S & 5 S RNAs are – 5, 000, 2, 000, 160 & 120 bases long respectively • 28 S, 5. 8 S & 5 S from same human pre-r. RNA – by nucleases at specific sites – 5 S r. RNA from separate precursor outside nucleolus Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Processing of r. RNA precursor • • Pre-r. RNA has lots of modified nucleotides

Processing of r. RNA precursor • • Pre-r. RNA 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 r. RNA interactions with other molecules Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

Processing of r. RNA precursor • Pulse-chase with 14 C-methionine – 45 S peaks

Processing of r. RNA precursor • Pulse-chase with 14 C-methionine – 45 S peaks in nucleolar material after 10 min – 32 S peaks in nucleolar material after 40 -150 min – 32 S converted to 28 S – other product, 18 S r. RNA, in cytoplasm within 40 min – After 2 or more hours, nearly all of radioactivity has left nucleolus & most has accumulated in cytoplasmic 28 S & 18 S r. RNAs – Radiolabel in in 4 S RNA peak of cytoplasm represents CH 3 groups transferred to small t. RNAs Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 13

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

Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 14

small, nucleolar RNAs (sno. RNAs) • packaged with proteins: sno. RNPs • sno. RNPs

small, nucleolar RNAs (sno. RNAs) • packaged with proteins: sno. RNPs • sno. RNPs associate with nascent r. RNA precursor • first to attach contains U 3 sno. RNA – binds to precursor 5' end for 5' end removal – U 3 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 3 E

small, nucleolar RNAs (sno. RNAs) • Other antisense sno. RNAs – encoded within intervening

small, nucleolar RNAs (sno. RNAs) • Other antisense sno. RNAs – encoded within intervening sequences of other genes – binds to specific portion of pre-r. RNA – required to modify a particular nucleotides – 200 different antisense sno. RNAs – one for each methylated or pseudouridylated site – Box C/D sno. RNAs - methylation – Box H/ACA sno. RNAs - pseudouridines Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 15

Ribosomal subunit assembly • Done in nucleolus • 2 protein types associate with r.

Ribosomal subunit assembly • Done in nucleolus • 2 protein types associate with r. RNA as it's processed – Proteins that remain in ribosomal subunits – proteins that have transient interaction with r. RNA • needed for processing • proteins that protect sites from cleavage Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

5 S r. RNA synthesis & processing (~120 bases long) • part of prokaryote

5 S r. RNA synthesis & processing (~120 bases long) • part of prokaryote & eukaryote large ribosomal subunit • In eukaryotes – 5 S r. RNA is encoded by large number of identical genes – separate from the other r. RNA 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 5 S r. RNA – 3' end removed during processing – 5 S r. RNA is transported to nucleolus – participates in ribosome subunit assembly Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

5 S r. RNA synthesis & processing • Polymerase III action – binds to

5 S r. RNA 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 3 E

Transfer RNAs • ~50 t. RNAs in plant & animal cells, • each encoded

Transfer RNAs • ~50 t. RNAs 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 t. RNA genes – nontranscribed spacers separate t. RNA genes Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E

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

Copyright, ©, 2002, John Wiley & Sons, Inc. , Karp/CELL & MOLECULAR BIOLOGY 3 E Figure 11. 16

Transfer RNAs • Transcribed by polymerase III – 1° transcript of t. RNA is

Transfer RNAs • Transcribed by polymerase III – 1° transcript of t. RNA 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 t. RNAs 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 3 E