Biol139-lecture29-20 - BIOL 139 Transcription Transcription Chapter 8 con’t Chapter pp 265 275 pp pp 331 344 igenetics pp Transcription

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Unformatted text preview: BIOL 139 Transcription Transcription Chapter 8 con’t Chapter pp 265 - 275 pp pp 331 - 344 igenetics pp Transcription Transcription • Transfer of information Transfer from dsDNA to a ssRNA dsDNA Process by which polymerization of ribonucleotides guided polymerization by complementary base pairing produces an RNA transcript of a gene of • no primer is necessary no • One strand of DNA is used (template-strand) One • Nucleotides are added in the 5’-to-3’ direction 5’-to-3’ • Uracil is incorporated in place of Thymine in RNA Uracil Transcription Process: Overview Transcription RNA-like strand • RNA polymerase (RNAP)- the enzyme which catalyzes RNA transcription • unwinding of DNA (transcription bubble) near gene unwinding before transcription can begin - prokaryotes RNAP does this Like Replication • initiation • elongation 3 stages: • termination Transcription: organization of a gene Transcription: • Genes are flanked by regions called Promoters Promoters Promoters – DNA sequences near the beginning of genes DNA that signal RNA polymerase where to begin transcription Terminators – Sequences in the RNA product that tells RNA polymerase where to stop (encoded by the DNA) RNA Negative Negative numbers Positive numbers The steps in transcription: Initiation Prokaryotes Initiation holoenzyme RNAP: large protein σ • exits as a holoenzyme holoenzyme core • core enzyme (multiple subunits) core • σ factor - initiation only σ factor factor • essential for promoter recognition recognition • increases affinity of RNA core for promoter region affinity • allows RNAP to find and bind tightly to promoter region bind • aligns RNAP at the +1 site (start site) aligns Promoters: • 2 important sequences part of promoter region • - 35 and -10 region (Pribnow box) 35 Initiation of RNA chains: Initiation • binding of RNAP holoenzyme to promoter region consensus 5’ TTGACA consensus TTGACA consensus 5’ TATAAT consensus TATAAT 1) RNAP holoenzyme binds loosely to -35 region (dsDNA), loosely then tightly to the -10 region of dsDNA (closed promoter) tightly -10 2) Localized unwinding of the two strands of DNA (≈ 17 bp around -10 region) creates transcription bubble, provides template strand for RNAP and exposes initiation site (+1) template (Open Promoter Complex now) 3) RNAP chooses correct strand to read (template) and aligns 1st two nucleotides (dinucleotide formed) RNA-like strand Template 4) phosphodiester bond forms between first 2 NTP’s of RNA chain 5) initiates ≈ 8-9 bp then σ factor is released • Core loses affinity for promoter region • initiation is over and elongation begins Elongation When core loses sigma factor it moves away from promoter • Transcription bubble ≈ 17 bp • Core completes elongation • RNAP unwinds dsDNA • DNA helix reforms and mRNA displaced from back The steps in transcription: Termination Termination rho Terminators • complementary region (G:C-rich) which form hairpin loop in the ssRNA – RNAP pauses on UUU and falls off. Termination mRNA 5’ C C C C G G G G G:C rich area in ssRNA is complementary and causes hairpin UUUU RNAP pauses on string of UUU region 3’ RNAP falls off and transcription ends Terminators exist either: • Intrinsic to RNA strand – intra-strand base pairing (rho-independent) Intrinsic • Extrinsic - requires an accessory protein - rho to stop (rho-dependent) Extrinsic rho Prokaryotes Amino acid region start stop Transcription in Eukaryotes Eukaryotes Promoter Promoter -120 -100 GC GC -80 -30 CCAAT Box TATA Box +1 site dsDNA Initiation of transcription TATA: positions transcription startpoint CAAT: regulates rate of transcription -initiation and how often GC: helps RNAP bind near startpoint mRNA made in the nucleus nucleus • usually differing lengths - called pre-RNA (primary transcript) pre-RNA In Eukaryotes : In 3 types of RNA polymerase: Pol I larger rRNAs for ribosomes Pol II mRNAs (structural genes for translation) Pol III tRNAs, 5S rRNA, and a few other small RNA species Processing begins immediately in nucleus: • mRNA is capped capped • mRNA is polyadenylated polyadenylated • mRNA has introns removed introns Process called post-transcriptional modification post-transcriptional Produces a mature messenger RNA (mRNA) Produces mature In Eukaryotes : In RNA processing after transcription produces a RNA mature messenger RNA (mRNA) mature 5’ Capping 5’ • Addition of methylated guanine cap at the 5’ end. methylated • Guanidine triphosphate added by a special capping Guanidine enzyme enzyme • Added in reverse orientation to the 5’ end after Added polymerization of the transcript’s first few nucleotides polymerization (20-30 NT) (20-30 • Methyl transferase then adds methyl group to the Methyl backward G backward • Critical for efficient translation of mRNA. The ends of eukaryotic mRNAs The *arrows indicate sites of methylation Features: • Triphosphate bridge Triphosphate • 5’ to 5’ linkage of guanine (reverse orientation) 5’ • Guanine is methylated 7’ position Guanine • First 2 NT’s of RNA can be methylated First This G is NOT encoded by the gene! This Essential for ribosome to bind to 5’-end Essential Eukaryotes : Polyadenylation Eukaryotes Polyadenylation • Addition of poly-A tail to 3’ end of mRNA (≈ 100-200 Adenosines) poly-A • NO DNA template for poly-A tail (no strings of T’s) NO 1) RNAP runs past end of gene (no termination sites) 1) 2) Ribonuclease cleaves the primary transcript to create a new 3’ end 2) 3) Poly(A)polymerase adds A’s onto this new 3’ end. Thought to stabilize mRNA from degradation Thought stabilize Aids in efficiency of translation Aids Embedded in transcript is a poly A site poly Cuts 11-30 NT downstream of poly A site to create new 3’ end Uses ATP In Eukaryotes : RNA splicing – removal of intron sequences R-loops which correspond to areas missing in mature RNA Pre-RNA or DNA Mature RNA Exons – amino acid coding regions. “Expressed sequences” found in Exons found both a gene’s DNA and in the mature mRNA Introns – non-amino acid coding regions. “Intervening sequences” Introns found in a gene’s DNA but not in the mature mRNA (removed from the primary transcript) 2.5 million base pairs Exons ≈ 50-2,000 Bp Introns ≈ 50-100,000 Bp Introns very common in Introns eukaryote genes eukaryote 14,000 nucleotides Mature mRNA How are Introns Removed? How • RNA primary transcript has all introns and exons RNA splicing involves: RNA • removal of introns • stitching together of exons to form contiguous RNA • very precise mechanism to protect integrity of reading frame! Must have mechanism in place to distinguish between intron and exon junction Must have enzymes to splice out the introns enzymes Splicing of introns is usually carried out by a complex of enzymes known as a spliceosome • spliceosome is a complex of proteins made up of snRNPs is complex (small nuclear ribonuclear proteins) Short sequences dictate the sites of splicing Short • Introns begin with 5’-GU and end with 3’-AG Introns • Also intron includes a branch-point sequence upstream of 3’ splice site Also • Key base is the Adenine of the branch site Key Splice Donor site Splice Acceptor site Regulated Process: snRNPs snRNPs • cut at junction of exon 1/ intron • loop intron and join to branch-point A • cut at junction of intron/exon 2 • Exons joined and lariat degraded In Prokaryotes : In 5’ end of transcript has a triphosphate, rather than a methylated cap No poly A tail at 3’ end No introns No post-transcriptional processing, 1ary transcript = mRNA 1ary In Eukaryotes : Post-transcriptional modifications: 1ary transcript undergoes 5’ cap 3’ poly A tail splicing to give mature mRNA mature ...
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This note was uploaded on 10/04/2011 for the course BIOL 139 taught by Professor Christinedupont during the Spring '10 term at Waterloo.

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