1. From Gene to Phenotype DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A CCAAACCGAGT U G G U U U G GC UC A Trp Phe Gly Ser Codon 3 5 3 5

2. Lecture Outline 11/4/05 The central dogma: –DNA->RNA->protein –Control of gene expression in prokaryotes vs eukaryotes Transcription –Initiation, Elongation,Termination mRNA processing –Introns and exons Other types of RNA

3. In prokaryotes –Transcription and translation occur simultaneously TRANSLATION TRANSCRIPTION DNA mRNA Ribosome Polypeptide Figure 17.3a

4. –RNA transcripts are modified before becoming true mRNA –Transcription and translation occur in separate compartments of the cell In Eukaryotes Figure 17.3b TRANSCRIPTION RNA PROCESSING TRANSLATION mRNA DNA Pre-mRNA Polypeptide Ribosome Nuclear envelope

5. One Gene -> One Enzyme EXPERIMENT Class I Mutants Class II Mutants Class III Mutants Wild type Minimal medium (MM) (control) MM + Ornithine MM + Citrulline MM + Arginine (control) Beadle and Tatum studied mutants of the bread mold Neurospora crassa and showed that each gene specified a particular enzyme

6. ArginineOrnithineCitrullinePrecursor Arginine is an essential amino acid, required for growth Normal cells can synthesisze arginine from precursors in the minimal medium Each mutant blocked a particular step of the pathway Specific enzymes (arrows) catalyze each step Which mutants can grow with which supplement? Mut 1Mut 2Mut 3 none000 Ornithine100 Citrulline110 Arginine111 “One Gene -> One Enzyme”

7. ArginineOrnithineCitrullinePrecursor Which mutants can grow with which supplement? Mut 1Mut 2Mut 3 none000 Ornithine100 Citrulline110 Arginine111 Mutant 2 can grow only if supplemented with citrulline or arginine Therefore mutant 2 must not make the enzyme to convert Ornithine to Citrulline

8. One strand of DNA is copied to make messenger RNA 5’... ATGAATGTC... 3’coding 3’... TACTTACAG... 5’template 5’... augaauguc->.. 3’RNA copy This is the “sense” or “coding” strand, because it reads the same as the mRNA This is the strand that is actually copied

9. Synthesis of an RNA Transcript –Initiation –Elongation –Termination Figure 17.7 Promoter Transcription unit RNA polymerase Start point 5353 3535 3535 5353 5353 3535 5353 3535 5 5 Rewound RNA transcript 3 3 Completed RNA transcript Unwound DNA RNA transcript Template strand of DNA DNA RNA polymerase binds to a promoter sequence mRNA copy of gene is synthesized 5’ to 3’ Termination sequence causes transcription to stop

10. RNA Polymerase Elongation RNA polymerase Non-template strand of DNA RNA nucleotides 3 end C A E G C A A U T A G G T T A A C G U A T C A T CCA A T T G G 3 5 5 Newly made RNA Direction of transcription (“downstream) Template strand of DNA

11. RNA synthesis RNA synthesis is similar to DNA synthesis except: –Does NOT need a primer –No proofreading Why not? mRNA has high turnover. An error in one molecule will not be inherited Both strands of DNA can serve as the template –Some genes are on one strand, other genes are on the other

13. See an animation from www.dnai.org

14. Initiation in eukaryotes TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide Eukaryotic promoters TATAA A A ATATTTT TATA box Start point Template DNA strand 5 3 3 5 Several transcription factors Additional transcription factors Transcription factors 5 3 3 5 1 2 3 Promoter 5 3 3 5 5 RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Several transcription factors must bind to promoter sequences upstream of the gene Then RNA polymerase can bind TATA box

15. RNA processing in eukaryotes A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end Protein-coding segment Polyadenylation signal Poly-A tail 3 UTR Stop codonStart codon 5 Cap 5 UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P PP 5 3 1. Add 5’ cap 2. Add poly A tail to 3’ end A modified GTP is added, backwards, on the 5’ end About 200 A’s added at 3’ end

16. RNA processing in eukaryotes TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide 5 Cap Exon Intron 1 5 30 31 Exon Intron 104105146 Exon 3 Poly-A tail Introns cut out and exons spliced together Coding segment 5 Cap 1 146 3 UTR 5 UTR Pre-mRNA mRNA 3. Splice out introns

17. Spliceosomes RNA transcript (pre-mRNA) Exon 1 Intron Exon 2 Other proteins Protein snRNA snRNPs Spliceosome components Cut-out intron mRNA Exon 1 Exon 2 5 5 5 1 2 3 Special “small nuclear RNA” molecules do the splicing Mature mRNA Pre- mRNA

18. Most Eukaryotic genes have introns 13 kb 6 exons 5 introns Example: Red/Green colorblindness Example: beta globin 3 exons 2 introns 1.6 kb

19. Alternative splicing Make different mRNAs (and proteins) from same gene by splicing out certain exons Cell-type specific RNA splicing

20. 2.4 Mb 260 kb intron Some have many introns and alternative forms Human Dystrophin gene

21. Correspondence between exons and protein domains Gene DNA Exon 1 Intron Exon 2IntronExon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide

22. Four types of RNA mRNA –Messenger RNA, encodes the amino acid sequence of a polypeptide rRNA –Ribosomal RNA, forms complexes with protein called ribosomes, which translate mRNA to protein tRNA –Transfer RNA, transports amino acids to ribosomes during protein synthesis snRNA –Small nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing

23. 3 A C C A C G C U U A A G ACA C C U G C * G UGU C U GA G G U A A A G U C A G A C C C GA G A G G G G A C U C A U U U A G G C G 5 Hydrogen bonds Anticodon * * * * * * * * * * * RNA will fold to specific shapes Because RNA is single-stranded, parts of the molecule can base pair with other parts of the same molecule, causing it to fold into defined shapes. Some RNA molecules can even act as enzymes (ribozymes)

24. Summary