EGeneExpress - Gene Expression Molecular Genetics Molecular...

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Unformatted text preview: Gene Expression Molecular Genetics Molecular Genetics 2: • Replication – Precisely copying all the genetic information (DNA) – S-stage of cell cycle – Exact replicas passed to daughter cells Gene Expression • Gene Expression – – – – Using a specific bit of the genetic information Make a “working copy ” of the needed bit (gene) Take the working copy to the workshop (ribosome) Use the copied instructions to build a specific protein Feb 16, 2001 What is a gene? The Central Dogma of Biology • A unit of heredity (inherited information) • Structure: – Short DNA sequence of nucleotides – One chromosome carries hundreds of genes • Function: – Order of nucleotides in DNA determines order of amino acids in protein – Each gene codes for a different polypeptide DNA molecule Gene 2 Gene 1 Gene 3 Gene Expression • Copy the recipe from the master document (DNA gene) in the nucleus. • Use the copy of the recipe (mRNA) to produce the protein on ribosomes in the cytoplasm. Heyer RNA nucleotides can base pair with DNA nucleotides U pairs with A 1 Gene Expression Genes are Expressed as Proteins I.Transcription: DNA to RNA • 2 main stages 1) Transcription – DNA information copied to RNA – Occurs in the nucleus 1 • A specific gene is “turned on ” • Making RNA strand from DNA gene • Starts at Promoter - DNA region before a gene • Ends at Terminator - DNA region at the end of a gene 2) Translation – RNA information used to construct a protein – Occurs in the cytoplasm 2 3 The flow of genetic information Transcription of a gene Figure 17.7 The stages of transcription: initiation, elongation, and termination Promoter Transcription unit 5¢ 3¢ • RNA polymerase moves along DNA strand and builds RNA strand Start point 3¢ 5¢ DNA Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 1 RNA polymerase 5¢ 3¢ 3¢ 5¢ Template strand of DNA Unwound RNA DNA transcript 2 Rewound Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5¢ Æ 3 ¢. In the wake of transcription, the DNA strands re-form a double helix. DNA 5¢ 3¢ 3¢ 5¢ 3¢ 5¢ RNA transcript 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. 5¢ 3¢ 3¢ 5¢ 3¢ 5¢ Completed RNA transcript 2. Transcriptional Elongation 1. The initiation of transcription 1 Eukaryotic promoters 5¢ 3¢ in eukaryotic cells •Promoter TATA box 3¢ 5¢ 3 Additional transcription factors RNA polymerase II Figure 17.8 Heyer 3¢ 5¢ 3¢ 5¢ 3¢ Transcription factors 3¢ 5¢ 5¢ RNA transcript Transcription initiation complex C A 5¢ A T C CA AT 3¢ -end T U CA U G C A A TA GGT T A 5¢ Direction of transcription (“ downstream) Newly made RNA RNA-polymerase II 1. Moves 3 ’Æ5 ’ down template DNA 2. Un-zips 10 –20 DNA bases at a time 2 Several transcription factors Transcription factors • Proteins that bind to the promoter • Facilitate binding of the polymerase here • Catalyses the synthesis of RNA 5’Æ3 ’ • Starts moving “downstream ” from the start point RNA nucleotides RNA polymerase Start Template point DNA strand T U G •Transcriptional factors 3¢ 5¢ TA TA A A A ATA T T T T G • A region of ~25 bases “upstream ” from the gene • Contains the transcription start point • Often includes a “ TATA box” •RNA polymerase Non-template strand of DNA Promoter Transcription initiation complex Figure 17.7 A C G Template strand of DNA 3. Ribo-nucleotides align by base pairing with template DNA of gene at 3’ -end of elongating RNA strand 4. RNA synthesized 5 ’Æ 3’ (~60 nucleotides per sec) 5. RNA strand separates from DNA as polymerase passes 6. DNA re-zips Elongation Figure 17.7 2 Gene Expression 3. Termination (prokaryotes) 3. Termination (eukaryotes) Polyadenylation signal Protein-coding segment 5¢ 3¢ AAUAAA 5¢ UTR w Transcribed RNA forms “GC hairpin”. w Hairpin causes RNA poly to fall off, terminating transcription. * Each type produced by transcription from DNA . Types of RNA Start codon 3¢ UTR Stop codon Clip off transcript w RNA-poly transcribes past gene and past a “polyadenylation signal” w Enzyme recognizes the poly-a signal and clips off the RNA transcript. w RNA poly continues transcribing downstream for a ways before disconnecting. Processing of “primary transcript” RNA: addition of the 5¢ cap and the poly-A tail DNA TRANSCRIPTION RNA PROCESSING Pre-mRNA mRNA Ribosome TRANSLATION A modified methyl-guanine nucleotide added to the 5 ¢ end Although each RNA molecule has only a single polynucleotide chain, it is not a smooth linear structure. Within strand complementary base pairing : Regions of complementary AU or GC pairs allow the molecule to fold on itself forming helical structures called hairpin loops. Processing of “primary transcript” RNA: RNA splicing Protein-coding segment 5¢ 50 to 250 adenine nucleotides added to the 3 ¢ end by poly-A synthetase using ATPs Polypeptide Polyadenylation signal G PPP 5¢ Cap AAUAAA Start codon 5¢ UTR Stop codon 3¢ UTR 3¢ AAA…AAA Poly-A tail •Cap & tail protect mRNA from rapid degradation in the cytoplasm. •Eukaryotic mRNA stay active for hours, or even days, in the cytoplasm. •Prokaryotes lack cap & tail; mRNA only lasts for minutes. Figure 17.9 The roles of snRNPs and spliceosomes in pre-mRNA splicing 5¢ • Introns : “intervening sequences ” — non-coding regions of DNA within a gene • Must be excised from pre-mRNA before translation. 1 RNA transcript (pre-mRNA) Intron Exon 1 Exon 2 Protein Other proteins snRNA snRNPs 5¢ Exon Intron Pre-mRNA 5¢ Cap 30 31 1 Intron Exon Coding segment 104 Exon 105 146 2 5¢ Poly-A tail 146 UTR: Un-Translated Region outside of coding segment. Heyer Spliceosome Poly-A tail Introns cut out and Exons spliced together mRNA 5¢ Cap 1 3¢ UTR 3¢ 3¢ UTR Figure 17.10 The average eukaryotic pre-mRNA is only ~15% message Spliceosome components 3 5¢ mRNA Exon 1 Exon 2 Cut-out intron Figure 17.11 3 Gene Expression Correspondence between exons and protein domains Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation • Codons (“ words”) are RNA nucleotide triplets • Each codon represents a specific amino acid Domain 3 Domain 2 Domain 1 Polypeptide II. Translation: the sequence of mRNA codons determines the sequence of amino acids in the polypeptide Figure 17.12 Translation of RNA codons The Genetic Code II. Translation • On the ribosome • tRNAs translate the sequence of 3-base nucleotide “words” (codons) into a sequence of amino acids in a polypeptide • NOTE: the mRNA is not “turned into” protein! • • specific mRNA codons are associated with specific amino acids 64 codons , but only 20 aa’s – Many codons are redundant – Some are start/stop signals Transfer RNAs Carry Amino Acids An aminoacyl-tRNA synthetase joins a specific amino acid to a specific tRNA Aminoacyl-tRNA synthetase (enzyme) 1 Active site binds the amino acid and ATP. Amino acid P P PAdenosine ATP 2 ATP loses two P groups and joins amino acid as AMP. PyrophosphateP Pi Structure and symbol of transfer RNA • • Heyer tRNA molecules match amino acids to the appropriate codon tRNA anticodon - a triplet sequence on tRNA that base pairs with a mRNA codon P Adenosine Pi Pi Phosphates tRNA 3 Appropriate tRNA bonds covalently to amino acid, PAdenosine displacing AMP. AMP 4 Activated amino acid is released by the enzyme. Aminoacyl tRNA (an “activated amino acid ”) Figure 17.15 4 Gene Expression Ribosomes: the site of translation The Ribosome P site (Peptidyl -tRNA binding site) Ribosomes are bound to ER and free in cytoplasm A site (AminoacyltRNA binding site) E site (Exit site) E P Large subunit A mRNA binding site (b) Small subunit Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. Ribosome: Made of rRNA and protein Initiation of Translation Elongation 1) Next tRNA binds to A site 2) Adjacent amino acids linked 3) tRNA in P site leaves 4) Ribosome moves to next codon Process continues The initiation of translation • mRNA binds to ribosome at start codon [AUG] • First tRNA binds to mRNA ( anticodon to codon ) Polypeptide elongation Translation: Termination Fig. 7.10, 1-2 • When ribosome reaches stop codon, a releasing factor (protein) binds to the codon instead of a tRNA. • Polypeptide is released from tRNA. • Ribosome then released from mRNA. Heyer 5 Gene Expression Fig. 7.10, 3-4 Fig. 7.10, 7-8 Fig. 7.10, 9-10 Fig. 7.10, 11-12 Heyer Fig. 7.10, 5-6 Fig. 7.10, 13-14 6 Gene Expression Fig. 7.12 Making lots of protein • Many copies of mRNA can be made from one gene • Many ribosomes can make protein from the same mRNA • Amplification of information allows rapid production of proteins Not done yet! Protein Shape Determines Function Regulation of Gene Expression Critical to have the right protein at the right place and right time. • Post-translation modification • Specific 3-D shape • Shape is critical to function • Initiation of transcription* – “Transcription factors” • Post-transcriptional modification – Alternative splicing of mRNA — “RNA processing” • Translation • Post-translational modification* • Denaturation = loss of shape Ë loss of function Ribbon model of lysozyme protein Review Heyer * Major regulative processes (Topics for future lectures) Prokaryotes & Eukaryotes 7 Gene Expression Base-pair substitution Mutations Wild type • Single base changes: – Substitution changes mRNA Protein AUGA AGUUUGGCUA A 5¢ Met Lys Phe Gly Amino end one aa in protein chain 3¢ Stop Carboxyl end Base-pair substitution No effect on amino acid sequence – Deletion or Insertion leads to U instead of C AUG A AG UUUG GUU A A Frame Shifts; changes entire aa sequence downstream Met Lys Phe Missense Gly Stop A instead of G AUGAAGUUUAGUUAA Met Lys Phe Ser Stop Nonsense U instead of A AUGUAGUUUGGCUA A Met Types of mutations A missense point mutation: The molecular basis of sickle-cell disease Wild-type hemoglobin DNA 3¢ 5¢ C T G A T In the DNA, the mutant template strand has an A where the wild-type template has a T. A The mutant mRNA has a U instead of an A in one codon. 3¢ A 5¢ T C mRNA A G 3¢ Wild type mRNA 5¢ A UG A A GUU UGGCU A A Met Lys Gly Phe 3¢ Stop Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A U GU A A G U U U GG C U A mRNA 5¢ Base-pair insertion or deletion Protein Mutant hemoglobin DNA Figure 17.24 Stop U 5¢ 3¢ Met Stop Frameshift causing extensive missense U Missing AUGA AGUUGGCUA A Normal hemoglobin Glu Sickle-cell hemoglobin Val Met The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu ). Lys Leu Ala Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid A A G Missing A UGUU UGGCU A A Figure 17.23 Met Phe Gly Stop Figure 17.25 A problem of origins: which came first? Heyer 8 ...
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