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

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

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 rRNAgene rRNAgene rRNAgene DNA RNAs RNA pol I

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

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 7 methyl-G cap 5’ untranslated region Start Codon: AUG Stop Codon: UGA UAA UAG Poly A signal AAUAAA 3’ untranslated region A 200 3’ poly A tail Coding region; ORF (Open Reading Frame)

10. Structure of the mRNA cap

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

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

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 20210A mRNA 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. Intron 1 Overview of mRNA Splicing Exon 1 AGGU Exon 2 A AGG Consensus sequences A typical intron is 100 - 50,000 nucleotides long, starts with GU and ends with A(C,U) 17-37 AG. This adenosine is 17-37 nucleotides from splice site. A typical exon is 100 - 300 nucleotides long, starts with G and ends with AG.

17. Exon selection factor Exon selection factor U2 snRNPU1 snRNP Intron 1 Overview of mRNA Splicing Exon 1 AGGU Exon 2 A AGG Factors such as U1 and U2 snRNP identify splice sites Exons are identified by RNA sequences within the exons that are recognized by exon selection factors.

18. U2 U1 U2 snRNPU1 snRNP Intron 1 Overview of mRNA Splicing Exon 1 AGGU Exon 2 A AGG A U G G AA G G HO

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

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

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

22. Alternative splicing: Isozymes of Tropomyocin 1 234567 8 9 10 1112 There are two alternative poly- adenylation sites. The splicing pattern determines which one is used IntronsExons There are two alternative translation stop codons. The splicing pattern determines which one is used Netter 2891 Start of transcription Start of translation

23. 1 234567 8 9 10 1112 1 456 78 9 2 Exons 10 and 11 are spliced out in smooth muscle. Exon 3 is missing from smooth muscle tropomyosin mRNA. Splicing pattern used in smooth muscle Translation stops here. No exon 11, so polyadenylation occurs at the end of exon 12.

24. Two splicing patterns are used in striated muscle 1 234567 8 9 10 1112 1 3456 78 9 10 11 1 3456 78 9 10 12 If exon 11 is not removed by splicing, poly- adenylation occurs here, and exon 12 is lost. If splicing removes exon 11, its poly- adenylation site is removed. Polyadenylation occurs at the end of exon 12. Translation stops here. Exon 3, but not exon 2 is present in striated muscle tropomyosin mRNA A. B.