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

Lecture Outline 11/7/05 The central dogma: Various types of RNA 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)

Four types of RNA mRNA rRNA tRNA snRNA 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

RNA will fold to specific shapes 3 A C G U * 5 Hydrogen bonds Anticodon 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)

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

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

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 neb oys awt heb igc ate att heh otd og Function could be restored by an insertion nearby the neb oys aaw the big cat eat the hot dog

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

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?

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

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

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

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

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

Ribosomes Growing polypeptide tRNA molecules Large subunit Small 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

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

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

The initiation of translation Large ribosomal subunit P site 3 5 U A C Met Met 5 A U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Small ribosomal subunit mRNA binding site Translation initiation complex 1 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 2

Elongation tRNA with appropriate anticodon binds at A site 1 Amino end of polypeptide TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide E mRNA 3 Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP 2 GDP E E P P A 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

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

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

See the Animation www.dnai.org

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

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