11/8/11 Transcription to RNA From Gene to Phenotype DNA Gene 2 • The central dogma: molecule – DNA->RNA->protein Gene 1 – One Gene, One Enzyme Gene 3 • Beadle & Tatum expts. • Transcription DNA strand 3! 5! – Initiation (template) A C C A A A C C G A G T – Elongation – Termination TRANSCRIPTION U G G U U U G G C U C A • mRNA processing mRNA 5! 3! – Introns and exons Codon TRANSLATION • Other types of RNA 11/9/2011 Protein Trp Phe Gly Ser Amino acid In Prokaryotes transcription and translation occur simultaneously In Eukaryotes Nuclear – Transcription and envelope translation occur in separate compartments TRANSCRIPTION DNA DNA TRANSCRIPTION of the cell Pre-mRNA RNA PROCESSING mRNA Ribosome – RNA transcripts are mRNA TRANSLATION modified before becoming true mRNA Ribosome Polypeptide TRANSLATION Polypeptide Figure 17.3a Figure 17.3b One Gene -> One Enzyme “One Gene -> One Enzyme” EXPERIMENT Class I Class II Class III Wild type Mutants Mutants Mutants Minimal Normal bread-mold cells can medium synthesize arginine from precursors (MM) (control) in the minimal medium MM + Ornithine Mutant 2 could MM + grow if either Citrulline Precursor Ornithine Citrulline Arginine citruline or MM + arginine was Specific enzymes (arrows) Arginine is an essential Arginine amino acid, required for (control) supplied. catalyze each step growth Therefore it must lack the enzyme to make Citruline Precursor Ornithine Citrulline Arginine 1 11/8/11 From Gene to Phenotype RNA Polymerase Non-template strand of DNA DNA Gene 2 RNA nucleotides molecule Gene 1 Gene 3 T C C A A A T 3 C T U ! 3 end DNA strand 3! 5! ! G (template) A C C A A A C C G A G T T A U G G A 5 C A U C C A C TRANSCRIPTION ! A T A A G G T T U G G U U U G G C U C A mRNA 5! 3! Direction of transcription 5! Codon (“downstream) Template TRANSLATION strand of DNA Protein Trp Phe Gly Ser New RNA Amino acid Synthesis of an RNA Transcript DNA is copied to make messenger RNA Promoter Transcription unit 5! 3! 3! 5! RNA polymerase DNA This is the “non-template” Start point binds to a promoter RNA polymerase strand. Note that it reads – Initiation sequence the same as the mRNA 5! 3! 3! 5! Template strand of Unwound RNA DNA DNA transcript mRNA copy of gene is 5’ . ATGAATGTC . 3’ !! – Elongation synthesized 5’ to 3’ Rewound 3’ . TACTTACAG . 5’ !template! RNA 5! 3! 3! 3! 5! 5! RNA 5’ . augaauguc-> . 3’ !RNA copy transcript – Termination Termination sequence This is the template causes transcription to Like DNA, RNA elongates strand that is actually only at the 3’ end 5! 3! stop 3! 5! 5! 3! copied Completed RNA Figure 17.7 transcript Both strands of DNA can serve as the template Initiation in eukaryotes 1 Eukaryotic promoters Some genes are on one strand, other genes are on the other TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide TATA box Promoter 5! T A T A A A A 3! 3! A T A T T T T 5! TATA box Start point Template DNA strand 2 Several transcription factors Transcription Several transcription factors 5! 3! 3! 5! factors must bind to 3 Additional transcription promoter sequences factors upstream of the gene RNA polymerase II Transcription factors 5! 3! 5 Then RNA 3! ! 5! Small segment of human genome RNA transcript polymerase can bind Transcription initiation complex 2 11/8/11 RNA processing in eukaryotes RNA processing in eukaryotes 2. Add poly A 1. Add 5’ cap tail to 3’ end 3. Splice out introns 5! Exon Intron Exon Intron Exon A modified guanine nucleotide 50 to 250 adenine nucleotides 3! TRANSCRIPTION DNA Pre-mRNA 5! Cap Poly-A tail added to the 5! end added to the 3! end 1 30 31 104 105 146 TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA Introns cut out and RNA PROCESSING Pre-mRNA Protein-coding segment Polyadenylation signal mRNA Coding 5! exons spliced together 3 Ribosome segment mRNA ! G P P P AAUAAA AAA…AAA TRANSLATION Ribosome TRANSLATION Start codon Stop codon Polypeptide mRNA 5! Cap Poly-A tail 5! Cap 5! UTR 3! UTR Poly-A tail Polypeptide 1 146 5! UTR 3! UTR A modified About 200 A’s GTP is added, added at 3’ end backwards, on the 5’ end Spliceosomes Most Eukaryotic genes have Pre- mRNA RNA transcript (pre-mRNA) introns 5! Exon 1 Intron Exon 2 Protein 6 exons 5 introns 1 snRNA Other proteins Example: Red/Green colorblindness snRNPs Special “small Spliceosome nuclear RNA” molecules do 2 5! the splicing 13 kb Example: beta globin 3 exons 2 introns Spliceosome components Cut-out intron 3 mRNA 5! Mature mRNA Exon 1 Exon 2 1.6 kb Alternative splicing Some have many introns and many alternative forms Make different mRNAs (and proteins) from Human Dystrophin gene same gene by splicing out certain exons 260 kb intron 2.4 Mb Cell-type specific RNA splicing 3 11/8/11 Exons often correspond to particular RNA has many functions Functional Domains of a protein Gene DNA • 4 main types: Exon 1 Intron Exon 2 Intron Exon 3 – Messenger RNA Transcription – Ribosomal RNA RNA processing – Transfer RNA Translation – Small nuclear RNA Domain 3 • Folds to many possible shapes Domain 2 – Can act as an enzyme Domain 1 (“ribozyme”) SS RNA can base-pair with itself to form loops. Polypeptide DNA and RNA Compared Four types of RNA • mRNA 1. RNA is single stranded. – Messenger RNA, encodes the amino acid sequence of a polypeptide 2. Sugar is different. • rRNA 3. RNA uses U instead of T – Ribosomal RNA, forms complexes with protein called ribosomes, which translate mRNA to protein 4. RNA leaves nucleus, • tRNA DNA doesn’t. – Transfer RNA, transports amino acids to ribosomes during protein synthesis • snRNA – Small nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing 4 .