In this chapter, the nature of genetic material and the manner of its replication are revealed. The detail of how we know what we know is explained, and gives insight into the nature of scientific research and discovery. It is clear that we can view the secrets of nature only because we stand on the shoulders of many who have preceded us. The spiral of knowledge continues.
The nucleus of a cell is somewhat analogous to the office of an architect. In the form of DNA, the master plans for the molecules that are to be made by the cell are housed and protected in the nucleus. A disposable working copy of the plan, transcripted as a coded sequence complementary to the master plan in a molecule of messenger RNA, is sent to the construction site. The site is the ribosome, which has its own instruction set in the molecules of ribosomal RNA. Polypeptides, that may be either end products or enzymes needed to make the end product, are formed by the ribosome from amino acids delivered by molecules of transfer RNA. The process is not quite as straight forward in eukaryotes as it is in prokaryotes. However, the system of checks and balances that makes DNA replication work is remarkable.
While it's desirable to have all of the parts of an automobile in good working order, one would hardly want all of the components to be active at the same time. Nor would one want any one of the components, such as the horn, to be active all of the time. Too much of a good thing is not a good thing. This is also true of one's genome. There are times for genes to be active and times for them to be inactive. The ingenious mechanisms that have evolved to regulate the activity and inactivity of the genes are the subject of this chapter.
NUCLEIC ACIDS
INTRODUCTION Patterns of Heredity Explained by Chromosomes and Meiosis Enhanced the Study of Humans as Biological Organisms WHERE DO CELLS STORE HEREDITARY INFORMATION? Hammerling's Experiments with Acetabularia fig 14.1 Frog Nucleus Transplant Experiments Carrot Experiments WHICH COMPONENT OF THE NUCLEUS CONTAINS THE HEREDITARY INFORMATION? Genes Hold Hereditary Information The Griffith-Avery Experiments: Transforming Principle Is DNA The Hershey-Chase Experiment: Some Viruses Direct Their Heredity with DNA The Fraenkel-Conrat Experiment: Other Viruses Direct Their Heredity with RNA THE CHEMICAL NATURE OF NUCLEIC ACIDS Nucleic Acid First Isolated from Cell Nuclei Composed of Nucleotides (P.A. Levine) General structure fig 14.6 Numbering scheme for sugar structure fig 14.7 Nucleotides Strung Together in Chains Base Composition in Nucleotide Chains THE THREE-DIMENSIONAL STRUCTURE OF DNA Franklin's X-Ray Crystallography fig 14.9 Watson-Crick Analysis fig 14.11 HOW DNA REPLICATES Model Dependent on Complementarity of Strands Replication Is Semiconservative DNA replication model based on Meselson-Stahl experiments fig 14.15 Two Strands of DNA Are Replicated in Opposite Directions Replication begins at one or more origins of replication Actual replication occurs at Y shaped ends of replication fork fig 14.17 Replication occurs only in 5. to 3. direction Strands are elongated by different mechanisms Replication of leading strand, 5' to 3' strand Lagging strand, 3. to 5. strand replication Overall replication process is termed semidiscontinuous Comparing Prokaryotic and Eukaryotic DNA Replication Bacterial DNA double helix in form of single circle fig 14.18 Eukaryote DNA is not circular, but in chromosomes THE EUKARYOTIC CHROMOSOME Nucleus Contains a Large Amount of DNA fig 14.20 Histones Package DNA into Nucleosomes and Chromatin Euchromatin and Heterochromatin The Chromosome Further condensing occurs at beginning of mitosis Chromosome has centromere and telomeres at ends of DNA Full complement of chromosomes seen in karyotype fig 14.22 How Many Genes Are on a Chromosome? GENES: THE UNITS OF HEREDITARY INFORMATION Garrod Investigated Alkaptonuria, a Genetic Disorder The One Gene-One Enzyme Hypothesis How DNA Encodes Proteins
GENETIC EXPRESSION
INTRODUCTION Proteins Are Tools of Heredity Genes Code for Particular Polypeptides and Proteins CELLS USE RNA TO MAKE PROTEIN Polypeptides Assembled on Ribosomes in Cytoplasm fig 15.1 Cells Contain Three Classes of RNA Ribosomal RNA (rRNA) Transfer RNA (tRNA) Messenger RNA (mRNA) AN OVERVIEW OF GENE EXPRESSION Basic Apparatus of Gene Expression Shared by All Organisms fig 15.3 Transcription Translation fig 15.5 HOW GENES ENCODE INFORMATION Crick Determined Nature of Genetic Code Questioned Whether Code Was Simple or Punctuated In simple code, each nucleotide is part of a codon THE GENETIC CODE Deciphering the Genetic Code Examine process of translation in prokaryotes Initial portion of mRNA binds to rRNA in ribosome fig 15.7 Single mRNA codon exposed at polypeptide-making site tRNA with complementary anticodon binds to mRNA fig 15.8 Anticodon three nucleotides long Each tRNA specific for an amino acid Amino acid added to growing string of polypeptides Activating enzymes specify amino acid to be added to tRNA fig 15.9 Binds amino acid to tRNA One aminoacyl-tRNA synthetase enzyme for each amino acid Recognizes nucleotide-sequence information Recognizes protein-sequence information Code word is three nucleotides long Each recognizes different identities and numbers of tRNA's Special, non-amino acid associated codons Nonsense codons are stop signals: UAA, UAG, UGA AUG is the start signal Deviations From the "Universal Genetic Code" THE MECHANISM OF PROTEIN SYNTHESIS In Prokaryotes Synthesis Begins with Initiation Complex fig 15.10 Met-tRNA binds to small ribosomal subunit Initiation factors position met-tRNA Positioning critical to reading frame of mRNA Initiation complex binds to mRNA mRNA beginning marked by sequence complementary to rRNA on ribosome Allows base pairs to form between mRNA and rRNA Bacteria and eukaryotes differ in number of genes per mRNA transcript Several genes in one bacterial transcript (polycistronic) One gene per eukaryotic transcript Synthesis of Polypeptide Proceeds fig 15.11 Ribosome exposes codon adjacent to initiating AUG Appropriate tRNA briefly binds to its exposed mRNA site tRNA positioned by elongation factors Amino acid on tRNA adjacent to initial methionine The two amino acids chemically react with one another Methionine released from its tRNA Attached by peptide bond to adjacent amino acid Translocation occurs fig 15.12 Ribosome moves along mRNA to next codon Ejects prevoius tRNA from site Repositions tRNA with growing polypeptide Exposes next codon for incoming tRNA Process continues repeatedly from step B.2. Process stops when chain terminating code reached fig 15.13 No tRNA binds to nonsense codons Recognized by special release factors PROTEIN SYNTHESIS IN EUKARYOTES Slight Differences Between Prokaryotes and Eukaryotes tbl 15.2 Primary Difference in Eukaryotic Protein Synthesis Eukaryotic genes much longer than necessary Stretches of nucleotides cut out of mRNA transcript fig 15.14 Stretches called introns not translated Do not correspond to any portion of a polypeptide Exons are remaining, polypeptide specifying portions Exons are shorter than and scattered among introns RNA splicing cuts introns out of primary transcript "Processed" mRNA then translated
CONTROL OF GENE EXPRESSION
REGULATORY REGIONS DETERMINE GENE ACTIVITY Rationale for Controls Found in Bacteria Rationale for Controls Found in Multicellular Organisms GENERAL PRINCIPLES OF TRANSCRIPTIONAL CONTROL Gene Expression Is Regulated at Many Levels fig 16.2 Transcriptional control Post-transcriptional control Control Expression by Controlling RNA Polymerase Polymerase must have access to DNA helix Must be able to bind to gene's promoter Other sequences on DNA affect binding of polymerase and promoter HOW PROTEINS BIND TO SPECIFIC DNA SEQUENCES Molecular Recognition of Proteins to Regulatory Sequences The Helix-Turn-Helix Motif The Homeodomain Motif The Zinc Finger Motif The Leucine Zipper Motif TRANSCRIPTIONAL CONTROL IN BACTERIA Repressors Are "OFF" Switches Activators Are "ON" Switches Combination of Switches Sophisticated systems created by combining ON and OFF switches Example: lac operon of E. coli fig 16.13 TRANSCRIPTIONAL CONTROL IN EUKARYOTES Transcription Factors Assists binding of RNA polymerase to promotor fig 16.1 Several transcription factors provides numerous points for control Enhancers THE EFFECT OF CHROMOSOME STRUCTURE ON GENE REGULATION Histones Affect Gene Transcription Methylation Once Thought to Regulate Gene Transcription in Vertebrates POST-TRANSCRIPTIONAL CONTROL IN EUKARYOTES Gene Transcription Can Be Regulated at Points After Transcription fig 16.22 Processing of the Primary Transcript Transport of the Processed Transcript Out of the Nucleus
1. The cap and stalk died when the foot was removed. When the cap was cut off, the foot and stalk were not affected, and the cap regenerated. The A. mediterranea foot still grew an A. mediterranea cap, even with an A. crenulata stalk grafted onto it. Results suggested hereditary information resided in the foot (where the nucleus was).
2. They transplanted a nucleus into an enucleated fertilized egg with the resulting growth of a frog. An enucleated fertilized egg without a nucleus transplanted into it was not viable. Steward took macerated carrot tissue and managed to regenerate carrot tissue from single carrot cells.
3. The survivors were the ones injected with nonvirulent, uncoated bacteria or heat-killed, polysaccharide coated bacteria. The dead mice were injected with virulent bacteria containing a polysaccharide coat, or with a combination of live, non-polysaccharide-coated bacteria and dead, polysaccharide-coated bacteria. His conclusion was that the information specifying the coat from heat-killed bacteria was transferred to the live, non-polysaccharide-coated bacteria.
4. The primary component was DNA as evidenced by the fact that it was digested by DNAse.
5. Hershey and Chase labeled viral nucleic acid and viral protein coat with separate radioactive isotopes. After infected cells were analyzed, they found only viral nucleic acid inside the infected cells, not viral protein coat. Fraenkel-Conrat performed essentially the same experiment, creating hybrid plant viruses with nucleic acid from one species and viral protein coat from another. Plants developed lesions from these hybrid viruses, but only of the kind specified by the nucleic acid, not the viral protein coat.
6. A DNA molecule is composed of repeating units of deoxyribose, which is a five carbon sugar; a phosphate group; and a nitrogenous base. The three-dimensional shape of the molecule is a double helical strand. Hydrogen bonding between nitrogenous bases is responsible for holding the two strands of the helix together. Purines can only hydrogen-bond with pyrimidines, which is what accounts for equal numbers of purines and pyrimidines in a DNA molecule.
7. One strand of a DNA molecule is a "mirror" of the other. Meselson and Stahl showed that DNA replication was semiconservative by labeling new DNA with a heavy isotope so that older and newer strands could be identified. After allowing the DNA to replicate a while, it was spun on a cesium gradient, showing the newly-replicated material to weigh intermediate between the labeled and unlabeled DNA, suggesting that one chain (the new one) was "heavy," while the older (original) chain was "light."
8. The leading strand is replicated continuously while the lagging strand is replicated in fragments (Okazaki fragments). The reason for the discrepancy in replication strategy for the different strands is that the two strands are anti-parallel.
9. In bacteria, the duplex is nicked at one site, and a strand on one or both sides is displaced, creating one or two replication forks. These proceed around the circle until complete. Eukaryotic DNA has numerous replication forks along each chromosome, working in discrete units of 10,000 to 1,000,000 base pairs in length.
10. Histones are puck-shaped proteins around which the DNA duplex winds to form bead-shaped wads called nucleosomes. Nucleosomes are further condensed into visible dark areas in cell nuclei called nuclei. Chromatin is DNA: heterochromatin is usually densely packed and therefore not transcribed. Euchromatin, on the other hand, is DNA in a diffuse enough form to have its genes transcribed during interphase.
11. Beadle and Tatum wanted to test that hypothesis that DNA coded for specific enzymes, and tested their hypothesis on mold. They irradiated mold to see if they could create mutants incapable of synthesizing necessary enzymes in certain biosynthetic pathways. They were successful. Some of their mutants could not grow on a minimal medium unless they were supplied with the missing enzyme.
1. Ribosomal RNA (rRNA) functions in ribosomes to provide the site where a polypeptide is assembled. Transfer RNA (tRNA) transports amino acids to ribosomes to build a polypeptide. Messenger RNA (mRNA) passes from the nucleus to the cytoplasm to be a blueprint for protein synthesis, which is produced from chromosomal DNA information.
2. RNA polymerase initiates transcription and catalyzes the binding of the appropriate mRNA base to the DNA base being transcribed. RNA polymerase recognizes specific "start" and "stop" regions at the start of and end of a gene.
3. The sequences are read in a simple sequence with no separating or silent nucleotides. Crick and his colleagues chemically deleted a nucleotide to change the reading frame, then determined what further deletions were required to restore the proper reading frame. They found that the reading of the code remained nonsense when one or two additional deletions were made, but three deletions corrected the reading frame.
4. Each amino acid is coded for by a series of three nucleotides (codon). Artificial RNA molecules of a single sequence, i.e., UUU, were added to a mixture of RNA and protein from ruptured cells, and scientists observed the type of protein that resulted.
5. A codon is the three-nucleotide portion of the mRNA. An anticodon is the complementary three-nucleotide sequence on the tRNA. A nonsense codon does not code for an amino acid, but is rather interpreted by RNA machinery as instructions to "stop."
6. Only one codon at a time is receptive to a tRNA anticodon on the ribosome, and tRNA anticodons are specific for both the codons to which they bind and the amino acids they carry.
7. Translation is initiated through the formation of an initiation complex. Through elongation factors and translocation, the protein grows in length as the ribosome moves down the mRNA chain. When a "stop" nonsense codon is reached, release factors terminate translation and release the new polypeptide from the ribosome.
8. Elongation factors assist in the binding of the tRNA to the appropriate mRNA codon.
9. An intron is a segment of noncoding DNA. Exons are the segments of DNA that actually code for the production of a specific protein. Both introns and exons are transcribed into mRNA, but prior to leaving the nucleus, the introns are processed out of the so-called primary transcript, leaving only exons in the stretch of mRNA leaving the nucleus.
1. The major groove of DNA is the larger of the two grooves described by the double helix on its external surface. Inside the groove, hydrophobic methyl groups, hydrogen atoms, and hydrogen bond donors and acceptors can be accessed by regulatory proteins attached to the helix.
2. A helix-turn-helix is a regulatory protein consisting of two alpha helices joined by a short non-helical segment. It fits into the major groove of the DNA. Homeodomains function during the development of the organism as "developmental switches," and are critical in how different body regions are assembled. A zinc-finger is a regulatory protein typically containing both an alpha helix and a _-pleated sheet, as well as one to several atoms of zinc. A leucine zipper is a Y-shaped regulatory protein consisting of two interacting alpha-helix chains; the base of the "Y" consists of the helices being held together by their hydrophobic amino acids (usually leucine, hence the name).
3. A promoter is a sequence of nucleotides at the end of a gene that tells the RNA polymerase where to begin transcribing the gene. An operon is a cluster of genes transcribed as a unit. An operator is a regulatory site on a gene to which a repressor can bind, inhibiting transcription.
4. Repressors and activators are proteins that facilitate the inhibition or stimulation of transcription, respectively, by binding to specific regions of the gene (promoter sites or operator sites).
5. When bacteria are grown in a medium containing tryptophan, they do not need to manufacture the amino acid, so their trp gene is inhibited by a repressor which binds to the trp promoter. In the absence of tryptophan, the repressor protein is not triggered, does not bind to the promoter, and transcription of the trp gene occurs, supplying the amino acid to the bacterium.
6. cAMP participates in transcription control. When glucose levels are high in the surrounding medium, cAMP levels are correspondingly low, preventing CAP from binding to DNA.
7. When lactose is present in the environment, a lactose isomer binds to the lac repressor removing it from its regulatory position on the DNA and permitting transcription of the lac gene to occur.
8. Eukaryotic transcription factors stabilize and guide RNA polymerase. Enhancers in eukaryotic cells permit regulation of transcription from a distance, often a considerable distance, whereas bacterial regulation occurs directly at the site of the gene.
9. Methylation may serve to block accidental transcription of "turned off" genes.
10. The primary RNA transcript contains both introns and exons. Primary transcripts undergo processing prior to leaving the nucleus, during which the noncoding introns are removed.
11. mRNA can be affected several ways on its way from the nucleus to the ribosome: mRNA may or may not make it out through the nuclear membrane, some mRNAs may be translated over others, and some mRNA transcripts may be degraded prior to translation.