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The Molecular Biology of the Gene Identifying the Genetic Material • Mendel’s experiments—inherit chromosomes that contain genes • The Question now: – What are genes made of? • Scientists searching for the answer: – Griffith and Avery – Hershey and Chase Griffith-Avery Experiment: Transformation of Bacteria Controls THE STRUCTURE OF THE GENETIC MATERIAL •Experiments showed that DNA is the genetic material – The Hershey-Chase experiment showed that certain viruses reprogram host cells • To produce more viruses by injecting their DNA Head DNA Tail fiber 300,000 Tail Bacteriophage-virus that infects only bacteria Hershey-Chase Experiment: DNA, the Hereditary Material in Viruses Phage Radioactive protein Bacterium Empty protein shell Radioactivity in liquid Phage DNA DNA Batch 1 Radioactive protein Centrifuge Pellet 1 Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. 2 Agitate in a blender to separate phages outside the bacteria from the cells and their contents. 3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4 Measure the radioactivity in the pellet and the liquid. Radioactive DNA Batch 2 Radioactive DNA Centrifuge Pellet Figure 10.1B Radioactivity in pellet DNA and RNA are polymers of nucleotides •DNA is a nucleic acid – Made of long chains of nucleotide monomers Nucleotides of DNA • Nucleotides are the monomeric units that make up DNA 3 main parts: 5 carbon sugar—deoxyribose Phosphate group Nitrogenous base Nitrogenous bases of DNA • Pyrimidines: singlering structures Thymine (T) Cytosine (C) • Purines: larger, double-ring structures Adenine (A) Guanine (G) http://www.phschool.com/science/biology_place/biocoach/images/transcription/chembase.gif RNA • RNA is also a nucleic acid – But has a slightly different sugar – And has the pyrimidine, Uracil (U), instead of T http://www.phschool.com/science/biology_place/biocoach/images/transcription/chembase.gif Discovery of the Double Helix • 1953—James Watson and Francis Crick determined the structure of the DNA molecule to be a double helix – 2 strands of nucleotides twisted around each other Discovery of the Double Helix • Rosalind Franklin contributed to this discovery by producing an X-ray crystallographic picture of DNA – Determined helix was a uniform diameter and composed of 2 strands of stacked nucleotides – The structure of DNA • Consists of two polynucleotide strands wrapped around each other in a double helix Figure 10.3C Twist Double Helix Structure •Hydrogen bonds between bases – Hold the strands together •Each base pairs with a complementary partner – A base pairs with T – G base pairs with C Structure of DNA relates to its Function G C A T G C C G A T T C A A G T C G C C T A T A A G T G C G C G T A G C G C T A A A G T T C T A T A A T Structure of DNA is related to 2 primary functions: 1. Copy itself exactly for new cells that are created 2. Store and use information to direct cell activities DNA Complementary Strands • Strands run in opposite directions – Anti-parallel Complementary Strands of DNA • If one strand is known, the other strand can be determined 3’ 5’ A C G G T A T C C 5’ =T G C C =A =T =A G G 3’ DNA Replication •DNA replication depends on specific base pairing – DNA replication • Starts with the separation of DNA strands – Then enzymes use each strand as a template • To assemble new nucleotides into complementary strands A T A T A T A T A T C G C G C G C G C G G C G C G C G C T A T A T A T A T A A T A T A T Parental molecule of DNA C A Nucleotides Both parental strands serve as templates Two identical daughter molecules of DNA DNA Replication • Replication occurs simultaneously at many sites (replication bubbles) on a double helix Allows DNA replication to occur in a shorter period of time DNA Replication Process 1. Helicase unwinds the double helix to expose DNA nucleotides http://www.nature.com/nature/journal/v439/n7076/images/439542a-f1.2.jpg DNA Replication Process 2. Primase lays down an RNA primer to provide a 3’ OH group http://www.nature.com/nature/journal/v439/n7076/images/439542a-f1.2.jpg • • Can only add bases to the exposed 3’-OH group Therefore, DNA Replication always occurs in the 5’→ 3’ direction http://www.mun.ca/biochem/courses/3107/images/S tryer/Stryer_F31-23.jpg 3. DNA polymerase attaches complementary DNA nucleotides to the 3’ end of a growing daughter strand http://www.nature.com/nature/jour nal/v439/n7076/images/439542af1.2.jpg DNA Replication Process DNA Replication Process http://www.nature.com/nature/jour nal/v439/n7076/images/439542af1.2.jpg 4. DNA polymerase then removes the RNA primer and replaces it with complementary DNA nucleotides 5. DNA Ligase creates a covalent bond between the DNA fragments http://porpax.bio.miami.edu/~cmallery/150/chemistry/sf3x14a.jpg DNA Replication “Problem” • DNA Polymerase can only replicate in the 5’→ 3’ direction • One of the template strands would require replication in the 3’→ 5’ direction (WON’T WORK) • So, one daughter strand is made continuously while the other strand is made in short pieces called Okazaki fragments Overall Direction of Replication-toward the replication fork DNA Replication DNA Replication • Assures that daughter cells will carry the same genetic information as each other and as the parent cell. Each daughter DNA has one old strand of DNA and one new strand of DNA Semiconservative Replication Checking for Errors • 1/1,000,000,000 chance of an error in DNA replication – Can lead to mutations • DNA polymerases have a “proofreading” role – Can only add nucleotide to a growing strand if the previous nucleotide is correctly paired to its complementary base • If mistake happens, DNA polymerase backtracks, removes the incorrect nucleotide, and replaces it with the correct base Flow of Genetic Information • Flow of genetic information from DNA to RNA to protein • The DNA genetic code (genotype) is expressed as proteins which provide the physical traits (phenotype) of an organism GCTGCTAACGTCAGCTAGCTCGTAGC GCTAGCGCTTGCGTAGCTAAAGTCGA GCTCGCTTGCGTAGCTAAAGTCGAGC TGCTGCTAACGTCAGCTAGCTCGTAG AGCGCTTGCGTAGCTAAAGTCGAGCT AGCGCTTGCGTAGCTAAAGTCGAGCT GCTGCTAACGTCAGCTAGCTCGTAGC AGCGCTTGCGTAGCTAAAGTCGAGCT AGCGCTTGCGTAGCTAAAGTCGAGCT GCTGCTAACGTCAGCTAGCTCGTAGC AGCGCTTGCGTAGCTAAAGTCGAGCT AGCGCTTGCGTAGCTAAAGTCGAGCT GCTGCTAACGTCAGCTAGCTCGTAGC AGCGCTTGCGTAGCTAAAGTCGAGCT GCTGCTAACGTCAGCTAGCTCGTAGC AGCGCTTGCGTAGCTAAAGTCGAGC, cont. RNA Proteins Protein Synthesis • Transcription Process in which a molecule of DNA is copied into a complementary strand of RNA • Translation Process in which the message in RNA is made into a protein Forms of RNA 3 Main Types of RNA 1) mRNA (messenger RNA) – RNA that decodes DNA in nucleusbrings DNA message out of nucleus to the cytoplasm Each 3 bases on mRNA is a “codon” 2) tRNA (transfer RNA) – RNA that has the “anticodon” for mRNA’s codon The anticodon matches with the codon from mRNA to determine which amino acid joins the protein chain 3) rRNA (ribosomal RNA) – make up the ribosomes—RNA that lines up tRNA molecules with mRNA molecules Transcription produces genetic messages in the form of RNA RNA polymerase RNA nucleotide Direction of transcription Template strand of DNA Newly made RNA Figure 10.9A Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Transcription 1. Initiation: • RNA polymerase (enzyme) attaches to DNA at the promoter and “unzips” the two strands of DNA 2. Elongation: • RNA polymerase then “reads” the bases of DNA and builds a single strand of complementary RNA called messenger RNA (mRNA) 3. Termination: • When the enzyme reaches the terminator sequence, the RNA polymerase detaches from the RNA molecule and the gene Transcription The code on DNA tells how mRNA is put together. Example: DNAACCGTAACG mRNAUGGCAUUGC • Each set of 3 bases is called a triplet or codon (in mRNA) UGG CAU UGC • Eukaryotic RNA is processed before leaving the nucleus Noncoding segments called introns are spliced out • Coding segments called exons are bonded together • A 5’cap and a 3’ poly-A tail are added to the ends 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 Figure 10.10 Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Protein Synthesis • Transcription • Translation Process in which RNA is used to make a protein Transfer RNA molecules serve as interpreters during translation Amino acid attachment site • In the cytoplasm, a ribosome attaches to the mRNA and translates its message into a polypeptide • The process is aided by tRNAs Hydrogen bond RNA polynucleotide chain Anticodon Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Figure 10.11A Ribosomes build polypeptides Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation • The mRNA moves a codon at a time relative to the ribosome – A tRNA pairs with each codon, adding an amino acid to the growing polypeptide 1. Initiation: – – – Translation mRNA molecule binds to the small ribosomal subunit Initiator tRNA binds to the start codon (AUG— Methionine) in the P-site of the ribosome The large ribosomal subunit binds to the small one so that the initiator tRNA is in the P-site to create a functional ribosome 2. Elongation: – – – Codon recognition: anticodon of incoming tRNA molecule, carrying its amino acid, pairs with the mRNA codon in the A-site of the ribosome Peptide formation: polypeptide separates from the tRNA in the P site and attaches by a peptide bond to the amino acid carried by the tRNA in the A site Translocation: the tRNA in the P-site now leaves the ribosome, and the ribosome moves along the mRNA so that the tRNA in the A-site, carrying the growing polypeptide, is now in the Psite. Another tRNA is brought into the A-site Translation Translation 3. Termination: – – Elongation continues until a stop codon is reached— UAA, UAG, or UGA The completed polypeptide is released, the ribosome splits into its subunits • An exercise in translating the genetic code DNA RNA Start codon Polypeptide Stop codon Mutations • • Mutagenesis—creation of mutations Can result from Spontaneous Mutations • • Errors in DNA replication or recombination Mutagens—physical or chemical agents – High-energy radiation (X-rays, UV light) Types of Mutations • Mutations within a gene – Can be divided into two general categories. • Base substitution • Base deletion (or insertion) – Can result in changes in the amino acids in proteins. Normal hemoglobin DNA mRNA Mutant hemoglobin DNA Sickle-Cell Disease mRNA Normal hemoglobin Glu Sickle-cell hemoglobin Val Substitution Mutations • Missense mutation: altered codon still codes for an amino acid, although maybe not the right one • Nonsense mutation: altered codon is a stop codon and translation is terminated prematurely – Leads to nonfunctional proteins Insertions and Deletions • Frameshift mutation: addition or loss of one or more nucleotide pairs in a gene shifts the reading frame for translation and incorrect protein is made Are all Mutations Bad? • Although mutations are often harmful, – They are the source of the rich diversity of genes in the living world. – They contribute to the process of evolution by natural selection.