PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 13 Part 1 TRANSLATION OF mRNA

INTRODUCTION The translation of the mRNA codons into amino acid sequences leads to the synthesis of proteins A variety of cellular components play important roles in translation These include proteins, RNAs and small molecules 13-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Genes that encode polypeptides are termed structural genes These are transcribed into messenger RNA (mRNA) The translation of the mRNA codons into amino acid sequences leads to the synthesis of proteins The main function of the genetic material is to encode the production of cellular proteins In the correct cell, at the proper time, and in suitable amounts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.1 THE GENETIC BASIS FOR PROTEIN SYNTHESIS 13-3

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display He proposed that alkaptonuria was due to a missing enzyme, namely homogentisic acid oxidase Garrod also knew that alkaptonuria follows a recessive pattern of inheritance He proposed that a relationship exists between the inheritance of the trait and the inheritance of a defective enzyme He described the disease as an inborn error of metabolism Archibald Garrod 13-5

13-6 Metabolic pathway of phenylalanine metabolism and related genetic diseases Figure 13.1

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the early 1940s, George Beadle and Edward Tatum were also interested in the relationship among genes, enzymes and traits They specifically asked this question Is it One gene–one enzyme or one gene–many enzymes? Their genetic model was Neurospora crassa (a common bread mold) Their studies involved the analysis of simple nutritional requirements Beadle and Tatum’s Experiments 13-7

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Beadle and Tatum’s evidence for the one gene-one enzyme hypothesis

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-9

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Beadle and Tatum’s conclusion: A single gene controlled the synthesis of a single enzyme This was referred to as the one gene–one enzyme theory 1. Enzymes are only one category of proteins 2. Some proteins are composed of two or more different polypeptides The term polypeptide denotes structure The term protein denotes function Beadle and Tatum’s Experiments 13-10

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Translation involves an interpretation of one language into another In genetics, the nucleotide language of mRNA is translated into the amino acid language of proteins The genetic information is coded within mRNA in groups of three nucleotides known as codons The Genetic Code 13-12

13-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Special codons: AUG (which specifies methionine) = start codon AUG specifies additional methionines within the coding sequence UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted Refer to Table

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13-16 Figure 13.2 Figure 13.2 provides an overview of gene expression

The genetic code was deciphered in the early 1960s Thanks to several research groups, including two headed by Marshall Nirenberg and H. Gobind Khorana Nirenberg and his colleagues used a cell-free translation system Addition of synthetic RNA to DNase-treated extracts generates polypeptide synthesis Moreover, they added radiolabeled amino acids to these extracts Thus, the polypeptides would be radiolabeled and easy to detect Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Experiment 13A: Synthetic RNA Helped Decipher the Genetic Code 13-20

To make synthetic RNA, the enzyme polynucleotide phosphorylase was used In the presence of excess ribonucleoside diphosphates (NDPs), it catalyzes the covalent linkage of ribonucleotides into RNA Since it does not use a template, the order of nucleotides is random Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-21

An experimenter can control the amounts of nucleotides added For example, if 70% G and 30% U are mixed together, then … Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Codon PossibilitiesPercentage in the Random Polymer GGG0.7 x 0.7 x 0.7 = 0.34 = 34% GGU0.7 x 0.7 x 0.3 = 0.15 = 15% GUU0.7 x 0.3 x 0.3 = 0.06 = 6% UUU0.3 x 0.3 x 0.3 = 0.03 = 3% UGG0.3 x 0.7 x 0.7 = 0.15 = 15% UUG0.3 x 0.3 x 0.7 = 0.06 = 6% UGU0.3 x 0.7 x 0.3 = 0.06 = 6% GUG0.7 x 0.3 x 0.7 = 0.15 = 15% 

The Hypothesis The sequence of bases in RNA determines the incorporation of specific amino acids in the polypeptide The experiment aims to help decipher the relationship between base composition and particular amino acids Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure

13-23 Figure 13.3

The Data Radiolabeled Amino Acid Added Relative Amount of Radiolabeled Amino Acid Incorporated into Translated Polypeptide (% of total) Glycine49 Valine21 Tryptophan15 Cysteine6 Leucine6 Phenylalanine3 The other 14 amino acids 0 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Interpreting the Data Radiolabeled Amino Acid Added Relative Amount of Radiolabeled Amino Acid Incorporated into Translated Polypeptide (% of total) Glycine49 Valine21 Tryptophan15 Cysteine6 Leucine6 Phenylalanine3 The other 14 amino acids 0 Due to two codons: GGG (34%) and GGU (15%) Each is specified by a codon that has one guanine and two uracils (G + 2U) But the particular sequence for each of these amino acids cannot be distinguished Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Consistent with the results of an earlier experiment: A random polymer with only uracils encoded phenylalanine It is important to note that this is but one example of one type of experiment that helped decipher the genetic code

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the 1960s, Gobind Khorana and his collaborators developed a novel method to synthesize RNA They first created short RNAs (2 to 4 nucleotide long) that had a defined sequence These were then linked together enzymatically to create long copolymers They used these copolymers in a cell-free translation system like the one described in Figure 13.3 Refer to Table 13.5 RNA Copolymers Helped to Crack the Genetic Code 13-26

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Deciphering the code

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Nirenberg & Leder: Triplet Binding Assay

Triplet Binding Assay

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are four levels of structures in proteins 1. Primary 2. Secondary 3. Tertiary 4. Quaternary A protein’s primary structure is its amino acid sequence Refer to Figure 13.4 Levels of Structures in Proteins 13-28

13-29 Figure 13.4 The amino acid sequence of the enzyme lysozyme 129 amino acids long Within the cell, the protein will not be found in this linear state Rather, it will adapt a compact 3-D structure Indeed, this folding can begin during translation The progression from the primary to the 3-D structure is dictated by the amino acid sequence within the polypeptide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13-30 Figure 13.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are 20 amino acids that may be found in polypeptides Each contains a different side chain, or R group Nonpolar amino acids are hydrophobic They are often buried within the interior of a folded protein

13-31 Figure 13.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nonpolar and charged amino acids are hydrophilic They are more likely to be on the surface of the protein

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The primary structure of a protein folds to form regular, repeating shapes known as secondary structures There are two types of secondary structures  helix  sheet These are stabilized by the formation of hydrogen bonds Levels of Structures in Proteins 13-32

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings CH 2 OHOH O C OH CH 2 NH 3 + C -O-O CH 2 O SS CH CH 3 H3CH3C H3CH3C Hydrophobic interactions and van der Waals interactions Polypeptide backbone Hydrogen bond Ionic bond Disulfide bridge Protein Structure: Tertiary structure

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure This is the final conformation of proteins that are composed of a single polypeptide Proteins made up of two or more polypeptides have a quaternary structure This is formed when the various polypeptides associate together to make a functional protein Levels of Structures in Proteins 13-33

13-34 Figure 13.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A protein subunit

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13-37 A comparison of phenotype and genotype at the molecular, organismal and cellular levels Figure 13.7