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Start of Transcription

Start of Transcription. Point where transcription will start 5’…GpTpCpCpApCpGpTpCpApCpGpAp...3’ Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream

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Start of Transcription

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  1. Start of Transcription Point where transcription will start 5’…GpTpCpCpApCpGpTpCpApCpGpAp...3’ Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream DNA template strand Transcriptional initiation complex Start of Transcription 5’ pppA Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream DNA template strand (other strand not shown) riboATP OH 3'

  2. Elongation of a transcript Start of Transcription pppA Upstream ...CpApGpGpTpGpCpApGpTpGpCpTp... Downstream DNA template strand (other strand not shown) OH 3' RNA pppC-OH incoming nucleoside (CTP) triphosphate RNA polymerase Incorporated nucleotide pppApCpGpUpC ...CpApGpGpTpGpCpApGpTpGpCpTp.... H2O ppi 2pi pyrophosphate phosphate OH 3’ + Hydrolysis of pyrophosphate (by pyrophosphatase) is an important driving force.

  3. RNA polymerase I • Synthesis of • 18S rRNA • 5.8S rRNA • 28S rRNA (5S rRNA is synthesized by polIII)

  4. rRNA genes are located on the “stalk” regions of chromosomes 13, 14. 15. 21 & 22

  5. rRNA genes are located in tandem arrays RNA pol I RNAs DNA rRNAgene rRNAgene rRNAgene

  6. Introducing the concept of RNA processing rRNA Processing(occurs in the nucleolar regions of the nucleus) rRNA gene (DNA) Precursor RNA Mature RNAs RNA polymerase 1 RNA processing enzymes 3’ OH 5’ ppp 18S 5.8S 28S

  7. RNA polymerase II • Synthesis of mRNA (and some small RNAs)

  8. RNA polymerase III • Synthesis of small RNAs including • 5S rRNA • tRNAs • Located in the nucleus (not nucleolus) Details of pol III promoters are not essential for this course

  9. mRNA Structure Stop Codon: UGA UAA UAG Poly A signal AAUAAA 7 methyl-G cap Start Codon: AUG A200 5’ untranslated region 3’ untranslated region Coding region; ORF (Open Reading Frame) 3’ poly A tail

  10. Structure of the mRNA cap

  11. Overview of pre-mRNA processing Primary transcript: RNA capping: 7mG polyadenlyation signal 3’end cleavage: 7mG An polyadenylation: 7mG RNA splicing: 7mG An Transport through a nuclear pore complex to the cytoplasm Check by nonsense mediated decay pathway (RNA splicing may precede, occur at the same time as, or follow 3’ end formation)

  12. 3’ end Formation Stop Codon polyadenylation signal (e.g.AAUAAA) 3’ Processing complex 3’end cleavage Poly A addition 3’ untranslated region Last exon

  13. Mutations that affect 3’ end formation • Example: hyperprothrombinemia • Due to a G-to-A transition at position 20,210 • Mutation causes an increase in the amount of prothrombin • Associated with about a 3-fold increased risk of myocardial infarction • Present in about 2% of the European population but rare in non-caucasians

  14. Mutations that affect 3’ end formation • Example: hyperprothrombinemia • 20210 is the last nucleotide before the polyA tail • Due to increased 3’ end formation, cells with the 20210A allele produce more prothrombin mRNA than those with the 20210G allele • The 20210AmRNA has a longer half-life than 20210G mRNA.

  15. Mutations that affect 3’ end formation • Typically, if 3’end mutations have an effect, they reduce the amount of mRNA that is made. • Example: beta-plus thalassemia

  16. Overview of mRNA Splicing This adenosine is 17-37 nucleotides from splice site. A typical intron is 100 - 50,000 nucleotides long, starts with GU and ends with A(C,U)17-37AG. Exon 1 AGGU Intron 1 A AGG Exon 2 Consensus sequences A typical exon is 100 - 300 nucleotides long, starts with G and ends with AG.

  17. Overview of mRNA Splicing Factors such as U1 and U2 snRNP identify splice sites Exon selection factor Exon selection factor U1 snRNP U2 snRNP Exon 1 AGGU Intron 1 A AGG Exon 2 Exons are identified by RNA sequences within the exons that are recognized by exon selection factors.

  18. Overview of mRNA Splicing U1 snRNP U2 snRNP Exon 1 AGGU Intron 1 A AGG Exon 2 U U1 G U2 A HO G A A G G

  19. U U1 G U2 A HO G A A G G G G U2 A U1 A G G OH A G

  20. Detail of the 2’-5’ phospodiester formed during mRNA splicing 3’-5’ Exon 2 2’-5’ 3’-5’ G G A 2’-5’ A G G OH A G Intron

  21. G G U2 A U1 A G G OH A G G A G G + G A A G To cytoplasm degraded

  22. Netter 2891 Alternative splicing: Isozymes of Tropomyocin There are two alternative translation stop codons. The splicing pattern determines which one is used Start of transcription 8 1 9 11 12 2 3 4 5 6 7 10 There are two alternative poly- adenylation sites. The splicing pattern determines which one is used Start of translation Exons Introns

  23. Splicing pattern used in smooth muscle 8 1 9 11 12 2 3 4 5 6 7 10 Exon 3 is missing from smooth muscle tropomyosin mRNA. Translation stops here. 7 8 1 9 2 4 5 6 12 Exons 10 and 11 are spliced out in smooth muscle. No exon 11, so polyadenylation occurs at the end of exon 12.

  24. Two splicing patterns are used in striated muscle 8 1 9 11 12 2 3 4 5 6 7 10 Exon 3, but not exon 2 is present in striated muscle tropomyosin mRNA A. Translation stops here. 7 8 1 9 11 3 4 5 6 10 If exon 11 is not removed by splicing, poly- adenylation occurs here, and exon 12 is lost. B. Translation stops here. 7 8 12 1 9 3 4 5 6 10 If splicing removes exon 11, its poly- adenylation site is removed. Polyadenylation occurs at the end of exon 12.

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