2 From DNA to Proteins

1. 2 From DNA to Proteins I. Basic Unit of Life: The Cell A. Prokaryotes and Eukaryotes B. Macromolecules 1. Lipids 2. Polysaccharides 3. Proteins 4. Nucleic Acids II. Central Dogma of Molecular Biology III. DNA Replication A. Replication Process IV. Gene: Basic Unit of Genetic Information Genetic Code VI. Transcription A. Promoters and RNA polymerase B. RNA Processing VII. Translation A. Biotech Revolution: Translation in the Nucleus of Eukaryotic Cells? VIII. Regulation of Gene Expression A. Prokaryotic Gene Expression 1. The lacOperon {“operon” seen in text as cap and lower case} 2. The trpOperon B. Eukaryotic Gene Expression

2. The Cell • A. Basic unit of life; and all living organisms are composed of one or more cells. • B. Many scientific discoveries have contributed to modern cell biology: • 1. Cytology • 2. Biochemistry • 3.Genetics

3. C. Prokaryotes and Eukaryotes. Differences between prokaryotic and eukaryotic cells: a) A membrane-bound nucleus is in eukaryotic cells and not in prokaryotes. b) Eukaryotic cells contain membrane-bound organelles, while prokaryotes do not. Examples of organelles and other structures found in eukaryotic cells are: (1) Nucleus—location of DNA and chromosomes. (2) Endoplasmic reticulum—makes and modifies newly synthesized polypeptides; synthesizes lipids. (3) Golgi body (Dictyosome in plants)— modifies polypeptides, and sorts and ships proteins and lipids for either secretion or for use inside the cell. (4) Mitochondria—produces ATP (chemical form of energy cells use). (5) Chloroplasts—site of photosynthesis in plants and algae. (6) Vesicles—functions such as transport or storage, and digestion. (7) Ribosomes—helps assemble polypeptides during protein synthesis. (8) Cytoskeleton—confers shape to cells and aids in the internal organization of the cell, allows cells and parts of cells to move, and aids in the movement of internal structures. c) Prokaryotes have circular DNA molecules, while eukaryotes have linear chromosomes condensed with many proteins.

4. D. Macromolecules • Organelles and other structures of cells are made of polymers called “macromolecules”: • 1. Lipids. a) A broad group of hydrophobic (insoluble in water) organic compounds. b) Make up cellular membranes and define cells’ boundaries and organelles. c) Chemically diverse and play various roles in the cell: Triglycerides (Figure 2.2a) store energy; Phospholipids (Figure 2.2b) main component of membranes ; Glycolipids (e.g. component of membranes of nerve tissue); Steroids (e.g. cholesterol);Terpenes (e.g. a derivative of vitamin A). d) Fatty acids are the primary components of triglycerides and phospholipids: (1) Have a long, unbranched chain of carbon, hydrogen, and oxygen atoms. (2) Can have two conditions (Figure 2.3): (a) Saturated—no double bonds present in the carbon chain. The fatty acids pack very tightly, and saturated lipids are solid at room temperature (e.g. stearic acid is C18) (b) Unsaturated—at least one double bond present in the carbon chain. The chains do not pack tightly and are liquid at room temperature (e.g. Oleic acid C18 with one double bond; Linolenic acid C18 with 3 double bonds). Are liquid at room temperature

5. 2. Polysaccharides a) Made of repeating units called “simple sugars” or “monosaccharides” (Figure 2.4). b) Have two major functions: (1) Structure—cellulose (plants) and chitin (exoskeleton of insects). (2) Energy storage—Starch and glycogen. c) Made up of one monosaccharide or two monosaccharides that alternate, with examples of cellulose (composed of glucose monomers; Figure 2.5) and chitin (Figure 2.6). d) Monomers can also form disaccharides, with examples of lactose (glucose + galactose), maltose (glucose + glucose), and sucrose (glucose + fructose).

6. 4. Proteins a) Large organic compounds that determine many organismal characteristics. b) Proteins have different roles: Enzymes; Hormones; Antibodies; Transcription factors; Structure; Movement;Regulation of cellular processes. c) Protein-coding DNA sequences make up about 10% of the DNA in humans, with the rest being composed of sequences for various types of RNA, regulation, or nothing. d) Made of monomers called amino acids, of which there are twenty (Figure 2.7). e) Amino acids can form three-dimensional conformations that will ultimately determine how the protein will fold and function. f) Each amino acid has a similar structure: (1) (-NH3+) at one end. (2) A (COO-) group at one end. (3) A “C” atom in the middle, called the α (alpha) carbon.(4) A side group (called an R group) that determines its characteristics. g) There are four major chemical classifications of amino acids: (1) Nonpolar(10 amino acids) (2) Uncharged polar (5 amino acids) (3) Negatively charged (acidic) polar (2 amino acids). (4) Positively charged (basic) polar (3 amino acids) h) Peptide bonds link amino acids together (Figure 2.8) by linking the carboxyl group of one amino acid to the amino group on the next amino acid.

7. Nucleic Acids • - Function • - chemical composition: nucleobase, nucleoside, nucleotide (Fig 2.9) • - Polymers of nucleotides: direction of polymers 5’3’; types of chemical bonds • - DNA: double helix (symmetry) – Watson & Crick model (dimensions of double helix & polarity) (Fig. 2.10, 2.11, 2.12) • - RNA: single strand (Fig 2.13); structure (single strand and some short sequences along the polymer form double stranded regions which maintain RNA’s integrity); can undergo secondary and tertiary structure. • - Types of RNA • - rRNA: prokaryotes – 5S, 16S, & 23S; eukaryotes – 5S, 5.8S, 18S, & 28S

8. DNA ----------------RNA---------------Protein ---------------- DNA Replication -semiconservative (Fig. 2.18) & requires energy input (ATP) - In prokaryotes a) starts at single origin of replication then moves biderctional. b)Two replication forks moving in opposite direction (bidirectional)at each replication bubble (Fig 2.19) - In eukaryotes a) multiple origins of replication each replicating bidirectionally. b) each replication unit, called replicon, extends outward until it merges with another replicon until the chromosome is duplicated to two chromatids(Fig 2.19) - Enzymatic details involved in a replication fork in bacteria are shown in Fig 2.20)

9. Types of Genes • - RNA-encoding genes • - Protein-encoding genes: • a) structural genes – proteins have structural or enzymatic function. • b) regulatory genes – control the expression/activity of structural genes. • Genetic Code • - almost universal • - degenerate (Fig. 2.22) • - 64 codons: one start (AUG), three stop (UAA, UAG, UGA), & 60 for amino acids • - Correct protein is made only if mRNA is read in the correct reading frame (Fig 2.23)

10. Transcription - Several types of transcripts - Nucleotide sequences are referred to by upstream (before +1; start of transcript) and downstream (after the +1 nucleotide). - Details of transcription (Fig. 2.24 & 2.25) – for any gene, one DNA strand, known as template or antisense) is used as a template & transcribed while the other complementary DNA strand is known as the sense strand. - Promoters (Fig. 2.26) a) prokaryotic: ~ 30 nucleotides long & contain a Pribnow box at -10 & a -35 region. b) eukaryotic: - RNA polymerase I – transcribes 5.8S, 18S & 28S rRNA. - RNA polymerase II – transcribes genes encoding proteins & there are two types of promoter sequences for RNA polymerase II: (i) TATA box at -25 (transcription start point) (ii) CAAT box at -75 - RNA polymerase III – transcribes tRNA, 5S rRNA & small nuclear and cytoplasmicRNAa. Promoters may be at start or within the gene.

11. -Other proteins called “transcription factors” are also involved in initiation by increasing the ability of RNA polymerase to bind to the DNA molecule. - Regulatory proteins control transcription by interacting directly with RNA polymerase or by interacting with the transcription factors. - Short DNA sequences called “enhancers” can also influence transcription: a) Usually about fifty to 100 base pairs in length. b) Located upstream or downstream, sometimes thousands of base pairs from the gene. c) Most likely bind to regulatory proteins that also interact with RNA polymerase and transcription factors at the promoter region. d) The DNA can loop out to enable the enhancer to be involved. The looping may result from the interaction between the regulatory protein and proteins such as transcription factors and RNA polymerase (Figure 2.27).

12. - RNA Processing • 1. Newly made RNA is called “heterogeneous nuclear RNA” (hnRNA), and is modified in three ways before leaving the nucleus (Fig 2.28): a) 5′ cap structure—a modified guanine base is added to the 5′ end. b) 3′ poly-A tail—a string of adenine ribonucleotides is added to the 3′ end. c) Removal of noncoding sequences—noncoding sequences called “introns” are removed from the coding sequences, called the “exons.” d) The 5’cap and 3’ tail allow for three other things to occur: (i) Facilitate transport out of the nucleus. (ii) Protect the mRNA from degradation. (iii) Maintain stability for translation. 2. Other types of RNA molecules are also processed. a) Primary transcripts of ribosomal and transfer RNA are also processed: (i) Introns are spliced from tRNA, and both the 3’ and 5’ ends are processed. (ii) A large rRNA transcript is made and cut in a stepwise process to generate ribosomal NRA molecules.

13. Translation • - molecules involved; wobble at the 3rd base of the codon allows degeneracy and reduces the number of tRNA molecules needed. • - Steps: initiation, elongation, translocation, & termination (Fig 2.30, 2.31, 2.32 & Table 2.2) • - Differences between prokaryotes and eukaryotes.

14. Biotech Revolution: Translation in the Nucleus of Eukaryotic Cells? 1. It has been thought that proteins were only made in the cytoplasm. 2. Experiments have suggested that translation may also occur in the nucleus, because all of the necessary elements for translation appear to be present. 3. Experimental data suggests translation does occur in the nucleus, and between 40% and 45% of a eukaryotic cells’ protein synthesis may occur in the nucleus.

15. Regulation of Gene Expression • A. The initiation of transcription is a major control point in prokaryotes and eukaryotes. B. Eukaryotic regulation is much more complex than prokaryotes. C. In prokaryotes, the most common form of regulation is at of transcription initiation, but translational controls also exist to lower the rate of translation.

16. E. Regulation of Prokaryotic Gene Expression - Operon concept. - Example of regulation of genes involved in catabolism is the lactose operon (Fig 2.33 & 2.34) (i) Negative induction by lactose. Lactose is taken up then hydrolyzed to glucose and galactose. Glucose can then be used in respiration and energy production. (ii) The lacoperon is subject to positive regulation to enhance the rate of transcription of the 3 structural genes. - In the absence of glucose, the amount of a moleculae called “cyclic AMP” (cAMP) increases inside of the cell. - cAMP binds to a cAMP binding protein called catabolite activator protein (CAP) forming cAMP-CAP complex, which binds near the promoter region and touches the RNA polymerase enhancing its activity several folds. - When glucose is present, it inhibits adenylcyclase, the enzyme required to form cAMP. In turn cAMP levels are low and the cAMP-CAP doesn’t form. Thus the rate of transcription is not increased. This is known as glucose repression.

17. - An example of regulation of genes in anabolism is the trpOperon (Figure 2.35) (i) Regulates the production of the amino acid tryptophan. (ii) Consists of several elements: a) Promoter. b) Operator gene overlapping the promoter region. c) Five genes encoding enzymes that catalyze the last steps of tryptophan synthesis. (iii) Repressor is inactive unless tryptophan binds to it. The activated repressor attaches to the operator and blocks transcription. (iv) If there is no tryptophan, the repressor cannot bind and transcription occurs. (v) Genes are repressed to keep from too much tryptophan production.

18. Regulation of Eukaryotic Gene Expression 1. Much more intricate and variable than in prokaryotes. 2. Eukaryotic cells do the following things to regulate gene expression: a) Regulate transcription of genes. b) Control mRNA processing. c) Control transport of mRNA to the cytoplasm. d) Regulate the rate of translation. e) Control availability of mRNA. f) Protein processing.

19. 3. There are many reasons why eukaryotic gene regulation is so complex: a) Larger genome size—eukaryotic cells have more DNA than prokaryotes. Eukaryotic DNA also has many noncoding regions. b) Compartmentalization within the cell—eukaryotic cells contain membrane-bound organelles, while prokaryotes do not. This requires that proteins made in the cytoplasm be transported to organelles. c) More extensive transcript processing—processes such as RNA splicing are native to eukaryotic organisms and are not in prokaryotic organisms. d) Scattering of genes—genes are scattered around the genome and not in operons, and transcription of all the genes must be tightly coordinated. e) Regulation from a distance—enhancer and silencer sequences may be great distances from the gene they regulate, even thousands of bases. f) Cell and tissue-specific gene expression—specific sets of genes are activated and inactivated in different cell types, since not every cell needs to use every gene within the chromosomes.

20. The amount of a final product (usually a protein) from a gene is controlled at many levels within the cell (Figure 2.37): a) Transcriptional control: (1) There are more promoters and regulatory sequences in eukaryotic cells. (2) Transcription factors” interact with the promoter and RNA polymerase, forming the transcription initiation complex. • Gene-specific regulatory proteins bind to special DNA control sequences that contain regulatory protein binding sites. • (4) Control sequences can be adjacent to or distant from structural genes. (5) Individual or distant genes often must be regulated in scattered groups or networks, sometimes on different chromosomes. b) Regulation of RNA processing and transport out of the nucleus to the cytoplasm: - Different cell types may process the same mRNA differently, by a process called “alternative splicing,” to yield different proteins (Figure 2.38): (a) The pre-mRNA for the hormone calcitonin (non-processed mRNA) contains five introns separating six exons. (b) The transcript can be processed to generate calcitonin mRNA in thyroid cells or calcitonin gene-related peptide (CGRP) mRNA in hypothalamus cells.

21. c) Translational control: (1) Influences the final synthesis of a protein product (translation). (2) Translation is controlled by protein initiation factors and proteins that repress (inhibit) translation. (3) How quickly mRNA degrades influences when mRNA can be translated.

22. Posttranslational control: (1) The alteration of protein products after they are synthesized. (2) Various methods of alteration include: (a) Protein folding and assembly with other proteins after synthesis. (b) The removal of amino acids. (c) Cleavage of the molecule, which is called proteolysis. (d) Modification of the protein molecule. (i) Addition of a sugar is called glycosylation. (ii) Addition of a phosphate is called phosphorylation. (e) Importing proteins into organelles. (f) Protein degradation.