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MOLECULAR BIOLOGY OF THE GENE Chapter 12 pp. 211-232 Major Contributors to DNA Griffith Hershey and Chase Franklin and Wilkins Chargaff Watson and Crick 2 2 NUCLEOTIDE (Building blocks of Nucleic Acids) This is what a nucleotide would look like in reality……. NUCLEOTIDE (often identified by their bases) Here is how we could draw it….. P S B BASES (differs in each nucleotide) Adenine (A) Cytosine (C) Guanine (G) Uracil (U) Thymine (T) Purines = double ring Pyramidines = single ring Chargaff’s Rules The amounts of A, T, G, and C in DNA: Identical in identical twins Varies between individuals of a species Varies more from species to species In each species, there are equal amounts of: A & T G & C All this suggests DNA uses complementary base pairing to store genetic info Human chromosome estimated to contain, on average, 140 million base pairs 11 Watson and Crick Model Watson and Crick, 1953 Constructed a model of DNA Double-helix model is similar to a twisted ladder Sugar-phosphate backbones make up the sides Hydrogen-bonded Received bases make up the rungs a Nobel Prize in 1962 15 3 end P P P P P P • Therefore replication takes place in opposite directions 5 end P • Each strand of the double helix is oriented in the opposite direction, P Figure 10.5B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 3 end 5 end A model for DNA replication: the basic concept (layer 1) A T C G T A A T G C (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A model for DNA replication: the basic concept (layer 2) A T A T C G C G T A T A A T A T G C G C (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A model for DNA replication: the basic concept (layer 3) T A T A T A C G C G C T A T A T A A T A T A T G C G C G C G A T C G T A A C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings T (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. A model for DNA replication: the basic concept (layer 4) T A T A T A C G C G C T A T A T A A T A T A T G C G C G C G A T A T A T C G C G C G T A T A T A T A T A T C G C G C A G a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. DNA replication is the process of copying a DNA molecule. is semiconservative, with each strand of the original double helix (parental molecule) serving as a template (mold or model) for a new strand in a daughter molecule. Replication Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA replication: A closer look • DNA replication begins at specific sites Parental strand Origin of replication Daughter strand Bubble Two daughter DNA molecules Figure 10.5A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA REPLICATION http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/fr ee/0072437316/120076/micro04.swf::DNA%20Replication%2 0Fork Replication: Prokaryotic Prokaryotic Bacteria Replication have a single circular loop Replication moves around the circular DNA molecule in both directions Produces two identical circles Cell divides between circles, as fast as every 20 minutes 32 Replication: Prokaryotic vs. Eukaryotic Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. origin replication is complete replication is occurring in two directions a. Replication in prokaryotes replication fork replication bubble parental strand new DNA duplexes b. Replication in eukaryotes daughter strand 33 Replication Errors Genetic variations are the raw material for evolutionary change Mutation: A permanent (but unplanned) change in base-pair sequence Some due to errors in DNA replication Others are due to to DNA damage DNA repair enzymes are usually available to reverse most errors 34 Function of Genes Genes Specify Enzymes Beadle and Tatum: Experiments on fungus Neurospora crassa Proposed that each gene specifies the synthesis of one enzyme One-gene-one-enzyme hypothesis Genes Specify a Polypeptide A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide Suggests that genetic mutations cause changes in the primary structure of a protein 35 Overview of Protein Synthesis DNA ---------> RNA ------> Protein Transcription Translation DNA serves as a template to make RNA (transcription), which then carries the code for making a protein. The code is deciphered to make the protein (translation). Gene 1 Gene 3 DNA molecule Gene 2 DNA strand TRANSCRIPTION RNA Codon TRANSLATION Polypeptide Figure 10.7 Amino acid Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Types of RNA RNA is a polymer of RNA nucleotides RNA Nucleotides are of four types: Uracil, Adenine, Cytosine, and Guanine Uracil (U) replaces thymine (T) of DNA Types of RNA Messenger (mRNA) - Takes genetic message from DNA in nucleus to ribosomes in cytoplasm Ribosomal (rRNA) - Makes up ribosomes which read the message in mRNA Transfer (tRNA) - Transfers appropriate amino acid to ribosome when “instructed” 39 Structure of RNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G P G S U A P U base is uracil instead of thymine C S P A S P C S ribose one nucleotide 40 RNA vs. DNA structure 41 The genetic code • Virtually all organisms share the same genetic code Figure 10.8A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/transcripti on.swf Fig. 17-7a-1 Promoter Transcription Factors bind so RNA Polymerase knows where to go at TATA box 5 3 Start point RNA polymerase DNA 3 5 Fig. 17-7a-2 Promoter 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 Unwound DNA 3 5 RNA transcript Template strand of DNA Fig. 17-7a-3 Promoter 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 RNA transcript 3 5 Promoter 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 3 5 RNA transcript 3 Termination 5 3 3 5 5 Completed RNA transcript 3 Processing Messenger RNA Pre-mRNA, is composed of exons and introns. RNA splicing: Primary transcript consists of: Some segments that will not be expressed (introns) Segments that will be expressed (exons) Performed by spliceosome complexes in nucleoplasm Introns are excised Remaining exons are spliced back together Result is mature mRNA transcript 54 Eukaryotic RNA is processed before leaving the nucleus Modifications to ends of primary transcript: Cap of modified guanine on 5′ end The cap is a modified guanine (G) nucleotide Helps a ribosome where to attach when translation begins Poly-A tail of 150+ adenines on 3′ end Facilitates the transport of mRNA out of the nucleus Inhibits degradation of mRNA by hydrolytic enzymes. Figure 10.10 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence NUCLEUS CYTOPLASM RNA Splicing In prokaryotes, introns are removed by “selfsplicing”—that is, the intron itself has the capability of enzymatically splicing itself out of a pre-mRNA 55 Aminoacyl t-RNA synthetase adds amino acids to tRNA tRNA molecules have two binding sites One associates with the mRNA transcript The other associates with a specific amino acid Each of the 20 amino acids in proteins associates with one or more of 64 species of tRNA tRNA molecules come in 64 different kinds All very similar except that One end bears a specific triplet (of the 64 possible) called the anticodon Other end binds with a specific amino acid type tRNA synthetases attach correct amino acid to the correct tRNA molecule All tRNA molecules with a specific anticodon will always bind with the same amino acid Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The structure of transfer RNA (tRNA) 3 A Amino acid C attachment site C A C G C U U A A U C * C A C AG G G U G U * C * * U C * GA G G U A * A * * 5 G C G G A U U U A A G * * C U C * G C G A G A G G * C C A G A Hydrogen bonds C U A G Anticodon (a ) Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosomes Ribosomal Produced Combined RNA (rRNA): from a DNA template in the nucleolus with proteins into large and small ribosomal subunits A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA The E (for exit) site The P (for peptide) site, and The A (for amino acid) site 64 P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) Large subunit E P A mRNA binding site Small subunit (b) 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. This schematic ribosome will appear in later diagrams. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Steps in Translation: Initiation Components necessary for initiation are: Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors (special proteins that bring the above together) Initiator tRNA: Always has the UAC anticodon Always carries the amino acid methionine Capable of binding to the P site 66 Steps in Translation: Initiation Small ribosomal subunit attaches to mRNA transcript Beginning of transcript always has the START codon (AUG) Initiator tRNA (UAC) attaches to P site Large ribosomal subunit joins the small subunit 67 Steps in Translation: Elongation “Elongation” refers to the growth in length of the polypeptide RNA molecules bring their amino acid fares to the ribosome Ribosome reads a codon in the mRNA Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read Joins the ribosome at it’s A site Methionine of initiator is connected of 2nd tRNA by peptide bond to amino acid 70 Steps in Translation: Elongation Second tRNA moves to P site (translocation) Spent initiator moves to E site and exits Ribosome reads the next codon in the mRNA Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read Joins the ribosome at it’s A site Dipeptide on 2nd amino acid is of 3nd tRNA by peptide bond connected to amino acid 71 Codon is translated in a 5 to 3 direction. The third base in the code does not always show a complement between codon and anticodon = WOBBLE. Wobble still exists only between purines and pyrimidines though. Ribosomes build polypeptides Next amino acid to be added to polypeptide Growing polypeptide tRNA molecules P site A site Growing polypeptide Large subunit tRNA P mRNA binding site A mRNA Codons mRNA Small subunit Figure 10.12A-C Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings TRANSLATION (RNA is decoded) Steps in Translation: Termination Previous tRNA moves to P site Spent tRNA moves to E site and exits Ribosome reads the STOP codon at the end of the mRNA UAA, UAG, or UGA Does not code for an amino acid Polypeptide is released from last tRNA by release factor Ribosome releases mRNA and dissociates into subunits mRNA read by another ribosome 74 Polysome (Polyribosome) 35 36 Gene Mutations Point Mutations (Substitutions) •Missense •Nonsense Frameshift Mutations •Insertion •Deletion 37 27 Figure 17.23 The molecular basis of sickle-cell disease: a point mutation Wild-type hemoglobin DNA 3 Mutant hemoglobin DNA 5 C T T In the DNA, the 5 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 T C mRNA A mRNA G A A 5 G 3 U 5 Normal hemoglobin Glu Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 3 Sickle-cell hemoglobin Val The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Figure 17.24 Base-pair substitution Wild type mRNA A U G A A G U U U G G C U A A 5 Protein 3 Met Lys Phe Amino end Gly Stop Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C A U G A A G U U U G G U U A A Met Lys Missense Phe Gly Stop A instead of G A U G A A G U U U A G U U A A Met Lys Phe Ser Stop Nonsense U instead of A A U G U A G U U U G G C U A A Met Stop Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 17.25 Base-pair insertion or deletion Wild type mRNA Protein 5 A U G A A G U U U G G C U 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 G U A A G U U U G G C U A Met Stop Frameshift causing extensive missense U Missing A U G A A G U U G G C U A A Met Lys Leu Ala Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid A A G Missing A U G U U U G G C U A A Met Phe Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Gly Stop Figure 17.26 A summary of transcription and translation in a eukaryotic cell DNA TRANSCRIPTION 1 RNA is transcribed from a DNA template. 3 5 RNA transcript RNA polymerase RNA PROCESSING 2In eukaryotes, the RNA transcript (premRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM 3After leaving the nucleus, mRNA attaches to the ribosome. mRNA 4 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide Activated amino acid Ribosomal subunits 5 TRANSLATION 5 A succession of tRNAs E A AAA UGGUU UA U G Codon Ribosome Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings add their amino acids to Anticodonthe polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.)