1. Overview of Gene Expression

Transcription

1 Chapter 17: From Gene to 1. Overview of Gene Expression 2. Transcription 3. The Genetic Code 4. Translation 5. Mutations 1. Overview of Gene Expression Chapter Reading pp How are Genes related to? Genes are segments of that code for a particular protein (or RN molecule) the human genome contains ~3 billion base pairs (bps) and ~25,000 genes most genes encode proteins when we talk about genes we will focus on those that express proteins the gene products of some genes are RN molecules that play a variety of roles in cells 1

2 Condition 11/2/2015 EXPERIMENT RESULTS Classes of Neurospora crassa Growth: No growth: Wild-type Mutant cells cells growing cannot grow and dividing and divide Minimal medium Minimal medium (MM) (control) Wild type Class I mutants Class II mutants Class III mutants these classic experiments led to the one geneone enzyme hypothesis MM ornithine MM citrulline MM arginine (control) we now know that genes code for more than just enzymes Summary of results Gene (codes for enzyme) Gene Can grow with Can grow on Can grow only Require arginine or without any ornithine, on citrulline or to grow supplements citrulline, or arginine arginine Class I mutants Class II mutants Class III mutants (mutation in (mutation in (mutation in Wild type gene ) gene B) gene C) Precursor Precursor Precursor Precursor Enzyme Enzyme Enzyme Enzyme Beadle & Tatum, 1941 Srb & Horowitz, 1944 Gene B Ornithine Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C CONCLUSION rginine rginine rginine rginine Gene Expression The expression of most genes involves two distinct processes: 1) Transcription of a gene into RN RN is a nucleic acid very similar to (RN uses U instead of T ) this is essentially creating a photocopy of the gene occurs in the nucleus 2) Translation of the RN transcript into protein accomplished by ribosomes, in the cytoplasm Prokaryotes vs Eukaryotes Nuclear envelope RN TRNSCRIPTION RN PROCESSING Pre- TRNSCRIPTION TRNSLTION Ribosome TRNSLTION Ribosome Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell 2

3 template strand From to RN to C C C C G G T molecule T G G T T T G G C T C Gene 1 TRNSCRIPTION U G G U U U G G C U C Gene 2 TRNSLTION Codon Trp Phe Gly mino acid Ser Gene 3 gene expression ends at transcription for genes encoding RN gene products Comparison of & RN RN sugar = Ribose single-stranded, C, G & U (uracil) sugar = Deoxyribose double-stranded, C, G & T (thymine) 2. Transcription Chapter Reading pp

4 Stages of Transcription INITITION RN polymerase binds to the promoter of a gene, separates ELONGTION RN polymerase makes RN complementary to the template strand Promoter Transcription unit Start point RN polymerase 1 Initiation Unwound Rewound Nontemplate strand of Template strand of RN transcript 2 Elongation RN transcript 3 Termination TERMINTION RN polymerase stops transcribing when end of gene is reached Completed RN transcript Direction of transcription ( downstream ) TT box Transcription factors RN polymerase II T T T T T T T Promoter 1 eukaryotic promoter Start point Nontemplate strand 2 Several transcription factors bind to 3 Transcription initiation complex forms Transcription initiation complex Template strand Transcription factors RN transcript Initiation of Eukaryotic Transcription Eukaryotic promoters contain a short sequence called a TT box. With the help of an array of transcription factors, RN polymerase binds to the promoter and starts transcription. RN polymerase Nontemplate strand of RN nucleotides T C C C end C U C C T G G T T Newly made RN Direction of transcription Template strand of ELONGTION 4

5 Termination of Transcription RN polymerase released In self-termination, the transcription of terminator sequences cause the RN to fold, loosening the grip of RN polymerase on the. RN transcript released In enzyme-dependent termination, a termination enzyme pushes between RN polymerase and the, releasing the polymerase. C-G rich stem-loop Rho termination protein Rho protein moves along RN triggered by stem/loop structure in RN or termination factors such as the Rho protein (prokaryotes) Termination of transcription Primary RN Transcripts Newly made RN molecules in eukaryotes are called primary transcripts because they need to be processed before they can carry out their function: 1 o transcript G P P P Cap UTR -coding segment Start codon codon Polyadenylation signal U UTR Poly- tail Messenger () primary transcripts require the following modifications: a 5 cap a poly- tail splicing out of introns Pre- Codon numbers Cap Exon Intron 1 30 RN Splicing Exon Intron Introns cut out and exons spliced together Exon Poly- tail Cap UTR Coding segment Poly- tail UTR The coding region of the primary RN transcript contains intervening sequences called introns that need to be removed or spliced out. The regions that are retained are called exons which after splicing form a continuous coding region. 5

6 snrn Exon 1 RN transcript (pre-) snrnps Intron Exon 2 Spliceosome Other proteins How is Splicing Carried Out? Spliceosomes are structures made of protein and snrn Spliceosome components Exon 1 Exon 2 Cut-out intron snrn in spliceosome base pairs with ends of intron sequences resulting in their being spliced out Exons and Structure Gene Exon 1 Intron Exon 2 Intron Exon 3 Transcription RN processing Translation Exons tend to code for distinct substructures called domains in proteins. Domain 2 Polypeptide Domain 3 Domain 1 Exon shuffling is thought to be an evolutionary process by which new genes are made. Various Roles of RN Transcripts 1) messenger RN () RN copy of a gene that encodes a polypeptide 2) ribosomal RN (rrn) RN that is a structural component of ribosomes 3) transfer RN (trn) delivery of correct amino acids to ribosomes during translation For some genes, the end-product is the RN itself (rrn, trn) 6

7 3. The Genetic Code Chapter Reading pp How do Genes code for s? Recall that proteins are linear polymers made of 20 different amino acids. Genes need simply to encode the identity of each amino acid in a given protein! i.e., genes must be capable of encoding 20 different amino acids and their order in a protein although contains only 4 different nucleotides, this is more than sufficient to specify 20 different amino acids Basis of the Genetic Code Each amino acid in a protein is specified by 3 nucleotide sequences called codons each of the 20 amino acids is coded for by a unique set of codons: e.g. TG = methionine (start codon) GGN = glycine C or CG = glutamine there are 64 possible codon triplets (4 x 4 x 4) more than enough to encode 20 amino acids and the signal to stop or end the protein (TG, T or TG) 7

8 First base ( end of codon) Third base ( end of codon) 11/2/2015 The Genetic Code Second base U C G UUU UCU UU Phe UUC UCC UC Tyr UGU UGC Cys U C U Ser UU UC U UG Leu UUG UCG UG UGG Trp G CUU CCU CU CGU U His CUC CCC CC CGC C C Leu Pro rg CU CC C CG Gln CUG CCG CG CGG G UU CU U GU U sn Ser UC Ile CC C GC C Thr U C G Lys rg Met or UG CG G GG start G GUU GCU GU GGU U sp GUC GCC GC GGC C G Val la Gly GU GC G GG Glu GUG GCG GG GGG G If the sequence is: 5 -CTGCCTGGGCTG-3 3 -GTCGGCCCGTTTC-5 (transcription) The transcript is: 5 -CUGCCUGGGCUG-3 (translation) The polypeptide is: *Met-Pro-Gly-Gln (stop) all proteins begin w/met The Genetic Code is Universal Genes from one species can be expressed in another! (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene 4. Translation Chapter Reading pp. 83,

9 Polypeptide mino acids trn with amino acid attached Ribosome trn Overview of Translation Ribosomes facilitate the production of polypeptides by: 1) matching codons in with complementary anticodons in trn U G G U U U Codons C G G G C nticodon 2) catalyzing peptide bonds between amino acids carried by trns trn molecules Growing polypeptide Exit tunnel Ribosome Structure E P Large subunit Small subunit (a) Computer model of functioning ribosome P site (Peptidyl-tRN binding site) E site (Exit site) E P Exit tunnel site (minoacyltrn binding site) Large subunit mino end E Growing polypeptide Next amino acid to be added to polypeptide chain trn binding site Small subunit (b) Schematic model showing binding sites Codons (c) Schematic model with and trn mino acid attachment site Transfer RN (trn) trn anticodon will base pair with a complementary codon in mino acid attachment site Hydrogen bonds Hydrogen bonds nticodon (a) Two-dimensional structure nticodon (b) Three-dimensional structure G nticodon (c) Symbol used in this book 9

10 mino acid minoacyl-trn synthetase (enzyme) minoacyl-trn Synthetases P denosine P P P denosine TP P P i P i P i trn minoacyl-trn synthetase trn minoacyl trn ( charged trn ) P denosine MP mino acid Computer model Each trn is loaded with the correct amino acid due to the action of these enzymes. Initiation of Translation Large ribosomal subunit Initiator trn Start codon binding site U C U G GTP Small ribosomal subunit P i GDP P site E Translation initiation complex small ribosomal subunit aligns with start codon of initiator trn met and large subunit then join the complex Ribosome ready for next aminoacyl trn mino end of polypeptide E P site site GTP Elongation Cycle of Translation GDP P i E E P P GDP P i GTP E P 10

11 Termination of Translation Release factor Free polypeptide 2 GTP codon (UG, U, or UG) 2 GDP 2 P i When a stop codon is reached there is no trn with a complementary anticodon. Instead a release factor fits in the site and catalyzes the dissociation of all components. Incoming ribosomal subunits (a) Start of ( end) Growing polypeptides End of ( end) Completed polypeptide Multiple Ribosomes translate the same Ribosomes Referred to as polyribosomes. (b) 0.1 m Ensuring Proper Structure (pg. 83) Cap Polypeptide Correctly folded protein Hollow cylinder Chaperonin (fully assembled) The cap comes off, and the properly folded Steps of Chaperonin protein is ction: released. 1 n unfolded polypeptide enters the cylinder from one end. 2 The cap attaches, causing the 3 cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide. The cap comes off, and the properly folded protein is released. Chaperonins are large protein structures that assist newly made polypeptides to ensure they fold properly. capture improperly folded proteins inside its cavity, forcing them to unfold and hopefully refold correctly 11

12 1 SRP ER LUMEN Ribosome Signal peptide 2 Translation in the ER SRP receptor protein Translocation complex Signal peptide removed ER membrane 6 CYTOSOL s destined for the secretory pathway have a signal peptide at the N-terminus that targets the protein to the ER lumen. Gene Expression in Prokaryotes RN polymerase Transcription and translation are not segregated in prokaryotes. Since prokaryotic RN transcripts don t need to be processed, translation can begin before transcription is finished! RN polymerase Polyribosome Polypeptide (amino end) Polyribosome Direction of transcription Ribosome ( end) 0.25 m TRNSCRIPTION RN transcript RN PROCESSING CYTOPLSM Exon NUCLEUS RN polymerase RN transcript (pre-) Intron mino acid trn minoacyltrn synthetase MINO CID CTIVTION Summary of Gene Expression Growing polypeptide P E Ribosomal subunits minoacyl (charged) trn E Codon TRNSLTION nticodon Ribosome 12

13 5. Mutation Chapter Reading pp Mutations mutation is any change in sequence: change of one nucleotide to another insertion or deletion of nucleotides or fragments inversion or recombination of fragments What causes mutations? errors in replication or repair chemical mutagenesis high energy electromagnetic radiation UV light, X-rays, gamma rays Sickle-cell nemia Wild-type hemoglobin Sickle-cell hemoglobin Wild-type hemoglobin C T T G Mutant hemoglobin C T G T G G U Normal hemoglobin Glu Sickle-cell hemoglobin Val a single nucleotide change causes a single amino acid change resulting in a malformed protein 13

14 Types of Mutations Silent mutations: have no effect on amino acid specification Missense mutations: result in the change of a single amino acid Nonsense mutations: convert a codon specifying an amino acid to a stop codon results in premature truncation of a protein Insertion/deletion mutations: cause a shift in the reading frame of the gene all codons downstream of insertion/deletion will be incorrect Wild type Silent Mutations template strand T C T T C C C G T T T G G T T T G G C T mino end U G G U U U G G C U Met Lys Phe Gly Carboxyl end (a) Nucleotide-pair substitution: silent instead of G T C T T C C C T T T G G T T T G G T T U instead of C U G G U U U G G U U Met Lys Phe Gly Missense Mutations Wild type template strand T C T T C C C G T T T G G T T T G G C T mino end U G G U U U G G C U Met Lys Phe Gly Carboxyl end (a) Nucleotide-pair substitution: missense T instead of C T C T T C T C G T T T G G T T T G C T instead of G U G G U U U G C U Met Lys Phe Ser 14

15 Nonsense Mutations Wild type template strand T C T T C C C G T T T G G T T T G G C T mino end U G G U U U G G C U Met Lys Phe Gly Carboxyl end (a) Nucleotide-pair substitution: nonsense instead of T T instead of C T C T C C C G T T T G T G T T T G G C T U instead of U G U G U U U G G C U Met Frameshift Insertion/Deletion Wild type template strand T C T T C C C G T T T G G T T T G G C T U G G U U U G G C U mino end Met Lys Phe Gly Carboxyl end (b) Nucleotide-pair insertion or deletion: frameshift causing immediate nonsense Extra T C T T C C G G T T T G T G T T T G G C T Extra U U G U G U U U G G C U Met 1 nucleotide-pair insertion Can cause a premature stop codon Frameshift Insertion/Deletion Wild type template strand T C T T C C C G T T T G G T T T G G C T U G G U U U G G C U mino end Met Lys Phe Gly Carboxyl end (b) Nucleotide-pair insertion or deletion: frameshift causing extensive missense or extended shift in reading frame missing T C T T C C C G T T T G G T T G G C T U missing U G G U U G G C U Met Lys Leu la 1 nucleotide-pair deletion 15

16 Frameshift Insertion/Deletion Wild type template strand T C T T C C C G T T T G G T T T G G C T U G G U U U G G C U mino end Met Lys Phe Gly Carboxyl end (b) Nucleotide-pair insertion or deletion: no frameshift, but one amino acid missing or the addition/ deletion of a single amino acid. T T C missing T C C C G T T T G T T T G G C T G missing U G U U U G G C U Met Phe Gly 3 nucleotide-pair deletion Must be a multiple of 3! Key Terms for Chapter 17 primary transcript,, trn, rrn, snrn transcription, promoter, TT box, RN polymerase 5 cap, poly- tail, intron, exon, splicing, spliceosome rho protein, stem-loop, codon, anti-codon, translation aminoacyl trn synthetase, polyribosome, signal peptide, release factor, chaperonin mutation: substitution, deletion, insertion silent, missense, nonsense mutations reading frame, frameshift Relevant Chapter Questions 1-9, 11 16