Gene Expression: From Gene to Protein

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1 What is a Gene? Beadle and Tatum Experiment (1941) Gene Expression: From Gene to Protein Neurospora crassa Strain Wild type (control) Mold growth arge mutant argf mutant No growth Growth on MM + Nothing Ornithine Citrulline Arginino succinate Arginine argg mutant argh mutant How Do Genes Specify Enzymes? Overview: Transcription and Translation arge + gene argf + gene argg + gene argh + gene Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Precursor Ornithine Citrulline Argininosuccinate Arginine arge mutants blocked at this step argf mutants blocked at this step argg mutants blocked at this step argh mutants blocked at this step

2 Overview: Transcription and Translation Genetic Code Transcription Overview (1)! Begins as RNA polymerase binds to DNA Transcription Overview (2)! DNA double helix begins to unwind

3 Transcription Overview (3)! RNA polymerase adds RNA nucleotides sequentially according to the DNA template Transcription Overview (4)! Enzyme and completed RNA transcript release from DNA template Organization of a Gene Transcription: Initiation! Promoter Control sequence initiates transcription! Transcription unit Portion of gene that is copied into RNA! Terminator Signals the end of transcription of a gene

4 Transcription: Elongation Transcription: Termination Transcription in Eukaryotes: 1. Pre-mRNA Modifications DNA Pre-mRNA RNA-coding sequence Promoter Exon Intron Exon Intron Exon Cap 5' UTR 3' UTR Exon Intron Exon Intron Exon 5' UTR Transcription by RNA polymerase II. Addition of 5' cap soon after transcription begins. Addition of 3' poly(a) tail. 3' UTR mrna splicing: introns removed Transcription in Eukaryotes: 2. mrna Splicing Pre-mRNA 5' end of pre-mrna Exon Intron Exon 1 2 snrnps snrna Proteins Segment of pre-mrna with an intron 3' end of pre-mrna 2 SnRNPs bind to the intron by recognizing its boundary sequences and loop the intron out, bringing the two exons close together. The active spliceosome has now formed. mrna Protein-coding sequence Translation start Translation stop The spliceosome cleaves the intron at its beginning. The intron folds back on and bonds to itself.

5 Alternative mrna Splicing Translation Overview! α-tropomyosin in smooth and striated muscle trna Structure Aminoacyl-tRNA synthetase Amino acid binding site Amino acid 1 ATP and the amino acid bind to the aminoacyl-trna synthetase. The enzyme catalyzes the joining of the amino acid to AMP, with the loss of two phosphates. Phosphates ATP-binding site Anticodon binding site Aminoacyl-tRNA complex AA-AMP complex KEY AA-AMP = aminoacyl-amp AA-tRNA = aminoacyl-trna Anticodon 4 AA-tRNA is released, and the enzyme is ready to enter another reaction series. 3 The enzyme transfers the amino acid from AA-AMP to the trna, forming AA-tRNA. AMP is released. 2 The correct trna binds to the enzyme.

6 Ribosomes! Made of ribosomal RNA (rrna) and proteins Two subunits: large and small Translation: Initiation (1)! Initiator trna (Met-tRNA) binds to small subunit Translation: Initiation (2)! Complex binds to 5 cap of mrna, scans along mrna to find AUG start codon Translation: Initiation (3)! Large ribosomal subunit binds to complete initiation

7 Elongation Initiator trna Empty trna from E site 4 When translocation is complete, the empty trna in the E site is released and the cycle is ready to go again. 5' cap mrna Codons transferase 1 An aminoacyl-trna binds the A site. trna AminoacyltRNA Genetic Code Exit 3 The ribosome translocates along the mrna to the next codon, thereby bringing the trna with the growing polypeptide to the P site and moving the empty trna to the E site. Aminoacyl 2 transferase cleaves the amino acid from the P site trna and bonds it to the amino acid on the A site trna. Elongation Initiator trna Empty trna from E site 4 When translocation is complete, the empty trna in the E site is released and the cycle is ready to go again. 5' cap mrna Codons transferase 1 An aminoacyl-trna binds the A site. Elongation Initiator trna Empty trna from E site 4 When translocation is complete, the empty trna in the E site is released and the cycle is ready to go again. 5' cap mrna Codons transferase 1 An aminoacyl-trna binds the A site. trna AminoacyltRNA trna AminoacyltRNA Exit 3 The ribosome translocates along the mrna to the next codon, thereby bringing the trna with the growing polypeptide to the P site and moving the empty trna to the E site. Aminoacyl 2 transferase cleaves the amino acid from the P site trna and bonds it to the amino acid on the A site trna. Exit 3 The ribosome translocates along the mrna to the next codon, thereby bringing the trna with the growing polypeptide to the P site and moving the empty trna to the E site. Aminoacyl 2 transferase cleaves the amino acid from the P site trna and bonds it to the amino acid on the A site trna.

8 Elongation Initiator trna Empty trna from E site 4 When translocation is complete, the empty trna in the E site is released and the cycle is ready to go again. 5' cap mrna Codons transferase 1 An aminoacyl-trna binds the A site. trna AminoacyltRNA Translation: Termination (1)! Begins when A site reaches stop codon Exit 3 The ribosome translocates along the mrna to the next codon, thereby bringing the trna with the growing polypeptide to the P site and moving the empty trna to the E site. Aminoacyl 2 transferase cleaves the amino acid from the P site trna and bonds it to the amino acid on the A site trna. Translation: Termination (2)! Release factor (RF) or termination factor binds to A site Translation: Termination (3)! Polypeptide chain released from P site

9 Translation: Termination (4) Polysomes! Remaining parts of complex separated! Multiple ribosomes can simultaneously translate a single mrna Simultaneous Transcription and Translation Signal Mechanism in ER! Can occur in prokaryotes (no nuclear envelope)

10 Mutations: Missense Mutation! Changes one sense codon to one that specifies a different amino acid Sickle-Cell Disease! Caused by a single missense mutation Sickle Cell Anemia! A single adenine to Thymine transversion leads to a change in the amino acid composition (glu to val) in Hemoglobin! As a result, red blood cells start to sickle Mutations: Nonsense Mutation! Changes a sense codon to a stop codon

11 Duchenne Muscular Dystrophy! A single point mutation can result in an abbreviated protein if a regular codon is changed to a stop codon. In DMD, a structural muscle protein is non-functional. Mutations: Silent Mutation! Changes one sense codon to another sense codon that specifies the same amino acid Mutations: Frameshift Mutation! Base-pair insertion or deletion alters the reading frame after the point of the mutation Tay-Sachs! A neurodegenerative disorder that leads to death before the age of 4 is caused by a frame shift mutation in the hexosaminidase A gene on chromosome 15. A 4 base insertion within exon 11 is the cause of 80% of Tay- Sachs Ashkenazi Jews)

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