Transcription Individual DNA regions (genes) copied to mRNA One DNA strand is template Single-stranded RNA produced mRNA template strand template strand template strand template strand

Transcription Overview Un beau jour, je suis allé au marché pour acheter du pain. Il faisait chaud. Alors, j’ai acheté aussi un limonade. Il faisait chaud. 2

Transcription overview What do we call this strand? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA gene transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA

Transcription overview What enzyme makes RNA? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 4

Transcription overview What direction is mRNA made? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 5

Transcription overview What direction is the template strand read? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 5’ 3’ mRNA 6

Transcription overview Which strand does the mRNA look like? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA 3’ 5’ transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 5’ 3’ mRNA 7

Transcription overview How do we know where to start and stop? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA 3’ 5’ transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 5’ 3’ mRNA 8

Transcription overview How is the RNA actually made? Transcription overview RNA polymerase synthesizes RNA 5′→ 3′ Starts at promoter, ends at terminator “upstream” “downstream” DNA +1 start codon stop codon coding region promoter terminator transcription start codon stop codon mRNA 5′ coding region 3′ 5′ UTR 3′ UTR translation NH3 COOH protein

Eukaryotic transcription 3 RNA polymerases: RNA polymerase I – rRNA RNA polymerase II – mRNA RNA polymerase III – tRNA RNA polymerase II from yeast

Eukaryotic transcription RNAP II recognizes: TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences Transcription factors TFIIB TFIID +1 Sp1 hERRa1 CAAT GATA TATA box Enhancers

Eukaryotic transcription RNAP II recognizes: TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences +1

RNA processing in eukaryotes DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

5′ cap methylated guanine “backward” 5′ to 5′ linkage Not encoded in DNA Capping enzyme Recognition by ribosome 5′ AGACCUGACCAUACC

RNA processing in eukaryotes DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template mRNA stability …UGGCAGACCUGACCA 3′ …UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA

RNA processing in eukaryotes DNA promoter exons introns primary transcript (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing transcription unbroken coding sequence transport to cytoplasm for translation final mRNA

Splicing Most genes interrupted by introns Introns removed after transcription Exons spliced together 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence

Splicing snRNPs recognize exon-intron boundaries RNA + protein Cut and rejoin mRNA

Splicing RPE65 mRNA in nucleus: 21,000 nt (14 exons) AAAAAAAAA mature RPE65 mRNA in nucleus: 1,700 nt (8%)

Splicing Alternative splicing: >1 protein from one gene 27,000 human genes, but >100,000 proteins

Splicing Mutations affecting splicing can cause genetic disease: cystic fibrosis retinitis pigmentosa spinal muscular atrophy Prader-Willi syndrome Huntington disease spinocerebellar ataxia myotonic dystrophy Fragile-X syndrome Or produce genetic susceptibility to disease: lupus bipolar disorder schizophrenia myocardial infarction type I diabetes asthma cardiac hypertrophy multiple sclerosis autoimmune diseases elevated cholesterol

Gene expression summary Prokaryotes Eukaryotes DNA DNA transcription transcription mRNA pre-mRNA cytoplasm nucleus capping polyadenylation splicing directly translated (even before being completely transcribed) protein mature mRNA transport to cytoplasm translation cytoplasm protein

Ribosome Large ribonucleoprotein structure E. coli: 3 rRNAs, 52 proteins Two subunits: large and small large subunit RNA small subunit protein

Eukaryotic Translation How does the ribosome find the correct start codon? Small ribosome subunit binds 5′ cap Scans to first AUG start codon stop codon cap mRNA 5′ coding region AAAAAAAAA… 3′ 5′ UTR 3′ UTR

the Genetic Code After finding start codon, use the genetic code: Shown as mRNA 5′ → 3′ 26

Mechanics of Translation Translation requires: mature mRNA ribosome tRNAs amino acids accessory proteins

tRNA Small RNAs (74-95 nt) made by transcription Intramolecular base pairing Anticodon complementary to mRNA codon anticodon

tRNA “Charged” by specific aminoacyl tRNA synthetase

Initiation of Translation Small ribosome subunit binds at start codon Prokaryotes: Shine-Dalgarno sequence (RBS) Eukaryotes: binds cap, scans mRNA 5′ AUG GAU GGG

Initiation of Translation First tRNA (Met, anticodon CAU) joins complex 5' 3' Met UAC mRNA 5′ AUG GAU GGG

Initiation of Translation Large ribosomal subunit joins 5' 3' Met UAC mRNA 5′ AUG GAU GGG

Initiation of Translation P site holds tRNA with first aa A site open for next tRNA P A 5' 3' Met UAC mRNA 5′ AUG GAU GGG

Initiation of Translation

Elongation Next tRNA enters CUA P A UAC mRNA 5′ AUG GAU GGG Asp 3' 5' Met UAC mRNA 5′ AUG GAU GGG

Elongation Peptidyl transferase forms peptide bond Amino acid released from tRNA in P site Met Met Asp mRNA 5′ UAC 3' 5' CUA 3' 5' AUG GAU GGG

Elongation Ribosome translocates one codon First tRNA binds briefly in E site until translocation completes Met Asp 5' 3' UAC mRNA 5′ CUA 3' 5' AUG GAU GGG

Elongation Process repeats Next tRNA can then enter the empty A site 5' 3' Gly CCC Met Asp P A mRNA 5′ CUA 3' 5' AUG GAU GGG

Elongation

Termination Ribosome stops at stop codon No matching tRNA Release factor binds Met Val Leu Asp Gly Phe Val Lys Gly Leu Gln P A Asp Ile RF GUC 3' 5' UUG CAG UAG

Termination Translation complex dissociates UUG CAG UAG GUC RF Met Val Leu Asp Gly Phe Val Lys Gly Asp Leu Gln Ile UUG CAG UAG 5' 3' GUC RF

Polyribosomes Next ribosome starts as soon as start codon is available Growing polypeptide N RNA subunits released Ribosome 3' 5' AUG mRNA Stop 5' – 3' direction of ribosome movement C N Released polypeptide

Protein Synthesis Pathways Free ribosomes Ribosomes bound to RER