Nucleic Acids and Protein Synthesis Created By Prof. Gary F. Porter, Ph.D. Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved.

Table of Contents (hyperlinked) Introduction Nucleotides and Nucleosides 3. Laboratory Synthesis of Nucleosides and Nucleotides 4. Deoxyribonucleic Acid: DNA 5. RNA and Protein Synthesis 6. Determining the Base Sequence of DNA: The Chain-Terminating (Dideoxynucleotide) Method 7. Laboratory Synthesis of Oligonucleotides 8. The Polymerase Chain Reaction 9. Sequencing of the Human Genome: An Instruction Book for the Molecules of Life © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 1. Introduction Nucleic acids carry genetic information in cells DNA – deoxyribonucleic acids (2 deoxyribose) RNA – Ribonucleic acids (ribose) DNA has two strands, twisted ladder (α helix), is antiparallel © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 1. Introduction 23 pairs of chromosomes 30,000 to 35,000 genes One gene – one protein Chromosome 19 (to the right) Four Nitrogenous Bases Adenine Cytosine Thymine (Uracil in RNA) Guanine © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 2. Nucleotides and Nucleosides Nucleotide hydrolysis yields one of four heterocycles, one of two pentoses, and phosphate. Nucleoside hydrolysis yields heterocyclic base, and pentose. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 2. Nucleotides and Nucleosides Practice Problem 25.1 Write the structures of other tautomeric forms of adenine, guanine, cytosine, thymine, and uracil Other uses of nucleotides and nucleosides They are found in NADH, coenzyme A (enzymic cofactors). Cyclic AMP is a hormone. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 3. Laboratory Synthesis of Nucleosides and Nucleotides A basic sugar and base moiety are coupled together. Note Bz protective group © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 3. Laboratory Synthesis of Nucleosides and Nucleotides A heterocyclic molecule is added to a protected ribosylamine. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 3. Laboratory Synthesis of Nucleosides and Nucleotides A chlorinated heterocyclic nucleoside can be modified. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 3. Laboratory Synthesis of Nucleosides and Nucleotides A nucleoside is converted to a nucleotide. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 3. Laboratory Synthesis of Nucleosides and Nucleotides 3A. Medical Applications 6-Mercaptopurine is used to treat acute leukemia. Allopurinol is used to treat gout. Acyclovir is used to treat herpes virus. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 4. Deoxyribonucleic Acid: DNA 4A. Primary Structure Links occur on the 3’ OH and 5’ OH of the pentose Phosphate between the pentoses. Termini for the strands are 3’ OH and 5’ OH. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 4. Deoxyribonucleic Acid: DNA 4B. Secondary Structure Chargaff’s Rules (%G + %A) (%C + %T) %A/%T % G/%C © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 4. Deoxyribonucleic Acid: DNA 4B. Secondary Structure X-Ray Crystallography confirmed presence of the repeating units. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 4. Deoxyribonucleic Acid: DNA 4C Replication of DNA Involves three key ingredients: DNA template DNA product DNA polymerase © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis 5A. Messenger RNA Synthesis – Transcription Involves three key ingredients: DNA – Template, mRNA – product, and RNA Polymerase. DNA  RNA  protein © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis 5B. Ribosomes – rRNA Protein synthesis is catalyzed by ribosomes. Ribosomal parts include a 30S subunit – binding mediation site, and 50S subunit – catalytic site. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis 5C. Transfer RNA 20 tRNA exist One for each amino acid Make up of tRNA 70 to 90 monomeric units, and has four folds. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis The Genetic Code One AA per Codon Codon is a triplet Duplicates exist Primer AA is N-formylmethionine Chapter 25, P 1125 © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis 5D. The Genetic Code DNA is transcribed to an antisense DNA. Antisense DNA is transcribed to mRNA. DNA code = mRNA code mRNA is translated to Amino Acids / Protein. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis Translation Involves three key ingredients mRNA Template, tRNA, and Amino Acids/Protein product. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5. RNA and Protein Synthesis Practice Problem 25.10 Practice Problem 25.11 © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 6. Determining the Base Sequence of DNA: The Chain-Terminating (Dideoxynucleotide) Method Restriction Endonucleases Cleave double stranded DNA, and Are space sequence specific. AluI – cleaves AG/CT No sticky end EcoR1 – cleaves G/AATTC Has a sticky end Forms a palindrome segment. – AATT--- / / © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 6. Determining the Base Sequence of DNA: The Chain-Terminating (Dideoxynucleotide) Method 6A. DNA Sequencing by the Chain-Terminating (Dideoxynucleotide) Method Unnatural Nucleotides with Markers Are used as unnatural terminators The dye is attached to the heterocyclic ring. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 6. Determining the Base Sequence of DNA: The Chain-Terminating (Dideoxynucleotide) Method 6A. DNA Sequencing by the Chain-Terminating (Dideoxynucleotide) Method Gel Electrophoresis is earlier method, size & charge separation, and slower. Capillary Electrophoresis is current method, size separation, and faster. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 7. Laboratory Synthesis of Oligonucleotides Antisense Oligonucleotides bind to crucial sequences in viral DNA or RNA. Prevents viral replication are used as primers in synthetic DNA synthesis Automated synthesizers are used. These are similar in design to those used for protein synthesis. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 8. The Polymerase Chain Reaction Amplification of DNA Molecule Uses DNA polymerase and Thermal Cycler Device. Small amount of starting material (picogram) is used. One key use is to diagnose genetic-related diseases by amplifying and characterizing DNA sequences. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 8. The Polymerase Chain Reaction Thermophilic bacteria are used to speed up the reaction. The entire process can be done at elevated temperatures. © 2014 by John Wiley & Sons, Inc. All rights reserved.

© 2014 by John Wiley & Sons, Inc. All rights reserved. 9. Sequencing of the Human Genome: An Instruction Book for the Molecules of Life Human Genome Project All human genes (35,000) have been mapped out. Only a small % of the genes is understood. This research is useful for testing DNA for potential genetic disorders, engineering proteins based upon knowledge of identified protein, and synthesizing drugs from database enzymes. © 2014 by John Wiley & Sons, Inc. All rights reserved.