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Chapter 3. the Beginnings of Genomic Biology – Chapter 3. The Beginnings of Genomic Biology – Molecular Genetics Contents 3. The beginnings of Genomic Biology – molecular genetics 3.1. DNA is the Genetic Material 3.6.5. Translation initiation, elongation, and termnation 3.2. Watson & Crick – The structure of DNA 3.6.6. Protein Sorting in Eukaryotes 3.3. Chromosome structure 3.7. Regulation of Eukaryotic Gene Expression 3.3.1. Prokaryotic chromosome structure 3.7.1. Transcriptional Control 3.3.2. Eukaryotic chromosome structure 3.7.2. Pre-mRNA Processing Control 3.3.3. Heterochromatin & Euchromatin 3.4. DNA Replication 3.7.3. mRNA Transport from the Nucleus 3.4.1. DNA replication is semiconservative 3.7.4. Translational Control 3.4.2. DNA polymerases 3.7.5. Protein Processing Control 3.4.3. Initiation of replication 3.7.6. Degradation of mRNA Control 3.4.4. DNA replication is semidiscontinuous 3.7.7. Protein Degradation Control 3.4.5. DNA replication in Eukaryotes. 3.4.6. Replicating ends of chromosomes 3.5. Transcription 3.5.1. Cellular RNAs are transcribed from DNA 3.5.2. RNA polymerases catalyze transcription 3.5.3. Transcription in Prokaryotes 3.5.4. Transcription in Prokaryotes - Polycistronic mRNAs are produced from operons 3.5.5. Beyond Operons – Modification of expression in Prokaryotes 3.5.6. Transcriptions in Eukaryotes 3.5.7. Processing primary transcripts into mature mRNA 3.6. Translation 3.6.1. The Nature of Proteins 3.6.2. The Genetic Code 3.6.3. tRNA – The decoding molecule 3.6.4. Peptides are synthesized on Ribosomes CONCEPTS OF GENOMIC BIOLOGY Page 1 CHAPTER 3. THE BEGINNINGS OF GENOMIC BIOLOGY – MOLECULAR GENETICS (RETURN) 3.1. DNA IS THE GENETIC MATERIAL. (RETURN) As the development of classical genetics proceeded from Mendel in 1866 through the In 1928, a British scientist, early part of the 20th century the understanding Frederick Griffith, published his that Mendel’s factors that produced traits were work showing that live, rough, avirulent bacteria could be carried on chromosomes, and that there were transformed by a “principle” infinite ways that the genetic information from 2 found in dead, smooth, virulent parents could assort in each generation to bacteria into smooth, virulent produce the genetic variety demanded by bacteria. This meant that the Darwin’s theories on “origin of species” on which bacterial traits of rough versus natural selection acted. This gave rise to the Frederick Griffith smooth and avirulence versus study of gene behavior of more complex traits (1879-1941) viru- and an understanding of genes in populations. virulence were controlled by a substance that could carry the phenotype from dead to live cells. At the same time a quest for the material Griffith’s observations on Pneu-mococcus were inside a cell, perhaps a subcomponent of a controversial to say the least, and inspired a spirited chromosome, that carried the genetic instructions debate and much experimentation directed at proving to make organisms what they are was ongoing. whether the “transforming principle” was protein or nucleic acid, the two main components of chromosomes identified early in the 20th century, well CONCEPTS OF GENOMIC BIOLOGY Page 2 before Griffith’s experiments. This debate continued the label determined. The 35S-labeled protein was found until Oswald Avery and his colleagues, Colin MacLeod, outside the infected cells, while the 32P-labeled DNA and Maclyn McCarty published their work in 1944 was inside the E. coli, indicating that DNA carried the unequivocally showing that DNA was, in fact, Griffith’s information needed for viral infection. transforming principle. This completely revolutionized genetics and is considered the founding observation of molecular genetics. Figure 3.1. An electron micrograph of bacteriophage T2 (left), and a sketch showing the structures present in the virus (right). The head consists of a DNA molecule surrounded by proteins, while the core, sheath, and tail fibers are all made of Oswald T. Avery Colin MacLeod Maclyn McCarty protein. Only the DNA molecule enters the cell. In 1953, more evidence supporting DNA being the genetic material resulted from the work of Alfred Once it was established that DNA was the genetic Hershey and Martha Chase on E. coli infected with material carrying the instructions for life so to speak, bacteriophage T2. In their experiment, T2 proteins attention turned to the question of “How could a were labeled with the 35S radioisotope, and T2 DNA was molecule carry genetic information?” The key to that labeled with was labeled with the 32P radioisotope. Then became obvious with a detailed understanding of the the labeled viruses were mixed separately with the E. structure of the DNA molecule, which was developed by coli host, and after a short time, phage attachment was two scientists a Cambridge University, James Watson disrupted with a kitchen blender, and the location of and Francis Crick. CONCEPTS OF GENOMIC BIOLOGY Page 3 Rosalind Franklin a young x-ray crystallographer working in the laboratory of Maurice Wilkins at 3.2. WASON & CRICK – THE STRUCTURE OF DNA. Cambridge University used a technique known as x-ray (RETURN) diffraction to generate images of DNA molecules that showed that DNA had a helical structure with repeating The basic laboratory observations that lead to the structural elements every 0.34 nm and every 3.4 nm formulation of a structure for DNA did not involve along the axis of the molecule. biologists. Rather Irwin Chargaff, an analytical, organic chemist, and physicists, Rosalind Franklin and Maurice Wilkins made the laboratory observations that led to the solution of the structure of DNA. Chargaff determined that there were 4 different nitrogen bases found in DNA molecules; the purines, adenine (A) and guanine G), and the pyrimidines, cytosine (C) and thymine (T), and he purified DNA from a number of different sources so he could examine the quantitative relationships of A, T, G, and C. He con- Rosalind Franklin Maurice Wilkins cluded that in all DNA molecules, the mole-percentage of A was nearly equal to the mole-percentage of T, while the mole-percentage of G was nearly equal to the mole-percentage of C. Alternatively, you could state this as the mole-percentage of pyri-midine bases equaled the mole-percentage of purine bases. These observations became known as Chargaff’s rules. Figure 3.2. X-ray diffraction image of DNA molecule showing helical structure with repeat structural elements. CONCEPTS OF GENOMIC BIOLOGY Page 4 These astute observations allowed Watson and Crick The key elements of this structure are: to synthesize together a 3-dimentional structure of a • Double helical structure – each helix is made from DNA molecule with all of these essential features. This the alternating deoxyribose sugar and phosphate structure was published in 1953, and immediately groups derived from deoxynuclotides, which are the generated much excitement, culminating in a Nobel monomeric units that are used to make up Prize in Physiology and Medicine, in 1962 awarded to polymeric nucleic acid molecules. Each nucleotide Franklin, Wilkins, Watson, and Crick. in each chain consists of a nitrogen base of either the purine type (adenine or guanine) or the pyrimidine type (cytosine or thymidine) attached to the 1’-position of 2’-deoxyribose sugar, and a phosphate group, esterified by a phospho-ester bond to the 5’-position of the sugar. Figure 3.3. Watson & Crick’s DNA structure. Their model consisted of a double helicical structure with the sugars and phosphates making the two hlices on the outside of the structure. The sugars were held together by 3’-5’-phosphodiester bonds. The bases pair on the inside of the molecule with A always pairing with T, and G always pairing with C. This pairing leads to Chargaff’s observations about bases in DNA. Figure 3.4. Structures of purine and pyrimidine bases in DNA, and structure of 2’-deoxyribose sugar. CONCEPTS OF GENOMIC BIOLOGY Page 5 • The nucleotides are held together in sequence order Figure 3.5. The building bocks of nucleic along the length of the polynucleotide chain by 3’-5’- acids are nucleotides and nucleosides. Any phosphodiester bonds, and the strands demonstrate base together with a deoxyribose sugar forms a deoxyribonucleoside, while if the a polarity as the 5’-OH at one end of a polynuc- sugar is ribose a ribonucleoside is formed leotide strand is distinct from the 3’-OH at the other (not shown). Addition of a phosphate on end of the strand. Often, but not always, the 5’- the 5’ position of the sugar froms nucleotides from nucleosides. strand end will have a phosphate group attached. • Each of the 2 polynucleotide chains of the double helix are held together by hydrogen bonds beween the adenosines in one strand and the thymidines in the other strand, and between the guanosines in one strand hydrogen bonded to the cytosines in the other strand. Figure 3.6. Base pairing between A and T involves two hydrogen bonds, and pairing between G and C involves 3 Figure 3.7. Strand of DNA hydrogen bonds. This means that the forces holding strands showing the 3’,5’-phosphodi- together in G=C base pair-rich regions are stronger than in A=T ester bonds holding base pair-rich regions. nucleotides together. CONCEPTS OF GENOMIC BIOLOGY Page 6 In order to get a uniform diameter for the molecule and have proper alignment of the nucleotide pairs in the middle of the strands, the strands must be orient- ed in antiparallel fashion, i.e. with the strand polarity of each strand of the double helix going in the opposite direction (one strand is 3’-> 5’ whie the other is 5’ -> 3’). The truly elegant aspect of this solution to DNA structure produces a spacing of exactly 0.34 nm between nucleotide base pairs in the molecule, and there are 10 base-pairs per complete turn of the helix.
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