From Gene to Phenotype- part 2

1. DNA molecule Gene 2 Gene 1 Gene 3 DNA strand (template) 5 3 A C C A A A C C G A G T TRANSCRIPTION G U G G U G C A U U U C 5 mRNA 3 Codon TRANSLATION Gly Phe Protein Ser Trp Amino acid From Gene to Phenotype- part 2

2. Lecture Outline 11/7/05 • The central dogma: • DNA->RNA->protein • Various types of RNA • The genetic code • Mechanisms of translation • Initiation, Elongation,Termination Exam 3 is next Monday. It will cover mitosis and meiosis, DNA synthesis, transcription, translation, genetics of viruses. (chapters 12, 13, 16, 17, part of 18)

3. 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

4. 3 A C C 5 A C G C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C * A G U G * * G A G C Hydrogen bonds G C U A G * A * A C * U A G A 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)

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

6. Second mRNA base U C A G U UAU UUU UCU UGU Tyr Cys Phe UAC UUC UCC UGC C U Ser UUA UCA UAA Stop Stop UGA A Leu UAG UUG UCG Stop UGG Trp G CUU CCU U CAU CGU His CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln CUG CCG CAG CGG G Third mRNA base (3 end) First mRNA base (5 end) U AUU ACU AAU AGU Asn Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U GUU GCU GAU GGU Asp C GUC GCC GAC GGC G Val Ala Gly GUA GCA GAA GGA A GUG GCG GAG GGG G The genetic code Glu

7. The code is a triplet code • the new boy saw the big cat eat the hot dog • The first evidence for triplet code came from deletion experiments • the new boy saw the big cat eat the hot dog • the neb oys awt heb igc ate att heh otd og • Function could be restored by an insertion nearby • the new boy saw the big cat eat the hot dog • the neb oys aaw the big cat eat the hot dog

8. The code is a triplet code • . . . or by two more deletions to get it back into the correct “reading frame” • the new boy saw the big cat eat the hot dog • the neb oys awt heb igc ate att heh otd og • the neb osa wte big cat eat the hot dog

9. Deciphering the Code • Your book tells how people used synthetic mRNAs and in vitro translation to determine decipher the codons. • E.g. UUUUUUUU -> phenylalanine only • UCUCUCUCUCU -> mix of leucine and serine • Why is it a mixture?

10. The code is redundant Second mRNA base U C A G U UAU UUU UCU UGU • Several different codons encode the same amino acid Tyr Cys Phe UAC UUC UCC UGC C U Ser UCA UUA UAA Stop Stop UGA A Leu UAG UCG Stop UUG UGG Trp G CCU U CUU CAU CGU His CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln CCG CUG CAG CGG G Third mRNA base (3 end) First mRNA base (5 end) U AUU ACU AAU AGU Asn Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U GUU GCU GAU GGU Asp C GCC GAC GGC GUC G Val Ala Gly GUA GCA GAA GGA A GUG GCG GAG GGG G

11. The code is comma free • No punctuation between words. • Therefore deletions cause frameshifts • It does have start and stop signals, however. • Start: AUG • Stop: UAG, UAA, UGA

12. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA Translation: the basic concept

13. 3 A Amino acid attachment site C C 5 A C G C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C * A G U G * * G A G C Hydrogen bonds G C U A G * A * A C * U A G A Anticodon The structure of transfer RNA (tRNA)

14. Amino acid attachment site 5 3 Hydrogen bonds A A G 3 5 Anticodon Anticodon (c) (b) Three-dimensional structure Symbol used in this book tRNA

15. Ribosomes TRANSCRIPTION DNA mRNA Growing polypeptide Ribosome TRANSLATION Polypeptide tRNA molecules Large subunit E P A Small subunit RNA + protein. We now think that RNA catalyzes the reactions. 5 3 mRNA

16. EPA model of a ribosome P site (Peptide site) A site (Acceptor site) E site (Exit site) Large subunit E P A mRNA binding site Small subunit

17. New amino acids are added to the carboxyl end of the polypeptide Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA 3 mRNA Codons 5

18. The initiation of translation Large ribosomal subunit P site 3 5 U C A Met Met A 5 G 3 U Initiator tRNA GDP GTP E A mRNA 5 5 3 3 Start codon Small ribosomal subunit mRNA binding site Translation initiation complex Small subunit binds just upstream of start Special initiator methionine binds to the start codon (AUG) The large subunit can now bind to make the active ribosome 1 2

19. tRNA with appropriate anticodon binds at A site Elongation 1 Amino end of polypeptide DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide E mRNA 3 Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP GDP 2 E E P A P A 2 GDP 3 GTP Peptide bond formed. Growing chain is now at A Translocaton. Everything moves left one notch. Polypeptide is now at P. Empty tRNA leaves the E site. E P A

20. Termination Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) Stop codon has no matching tRNA 1 2 3 Release factor binds and separates the polypeptide from the tRNA The ribosome separates from mRNA

21. Active site binds the amino acid and ATP. 4 1 3 An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA Amino acid Aminoacyl-tRNA synthetase (enzyme) P P P Adenosine ATP Adenosine P Pyrophosphate P Pi Pi Pi tRNA Appropriate tRNA bonds to amino Acid, displacing AMP. Adenosine P AMP Activated amino acid is released by the enzyme.

22. See the Animation • www.dnai.org

23. Inhibition of protein synthesis NOTE: Prokaryotes (this generally includes protein synthesis in mitochondria and chloroplasts)

24. DNA TRANSCRIPTION RNA is transcribed from a DNA template. 5 2 1 3 4 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) C A U A E A C Anticodon A A A U G G U G U U U A Codon Ribosome Summary of transcription and translation in a eukaryotic cell