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| homopolymers or non-repetitive heteropolymers |
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| What is nature of genetic material? |
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Chromosomes made of nucleic acids and proteins.
Originally assumed genes would be composed of amino acids because it was thought that they appeared to be the only biomolecules with sufficient complexity to convey genetic information.
Idea of DNA as genetic material emerged from studies on a pneumonia causing bacterium. Work of Frederick Griffith in 1928 |
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| Griffith’s Experiments with Streptococcus pneumoniae |
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Definition
R: Rough strains: non-pathogenic
S: Smooth stains: pathogenic
Non-virulent R strains gained virulence when mixed with heat-killed S virulent strains.
S gene codes for enzyme involved in synthesis of carbohydrate capsule.
Capsule confers virulence on S strains
Process by which R strains were transformed into S by heat killed extracts of virulent S was named Transformation by Griffith. |
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| DNA can carry genetic specificity |
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Bacteriophage: virus that infects a bacterial host.
1st step: Adsorption 2nd step: penetration
3rd step: replication 4th step: release of virions.
T2virus: DNA core + protein coat called capsid
Label Proteins with S35; Label DNA with P32 |
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| Which label(s) go in & appear in progeny |
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Much of labeled nucleic acid detected in progeny phage, while very little protein enters bacterial host.
If infected bacteria were violently agitated after entrance of DNA; protein shells (ghosts) were shaken off without affecting the ability of bacteria to produce new phage particles. Result with animal virion? |
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| 1938: Astbury-first X ray diffraction of DNA |
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1950s Wilkins & Franklin: confirmed DNA helical: see crossways pattern
Also, composed of more than one strand: 2 or 3 |
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| 1952: Alexander Todd lab showed DNA chains held together by 3’ 5’ phosphodiester bonds. |
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3’5’ phophodiester linkages
Removal of water between the 3’ OH and the 5’ phosphate of next nucleotide.
Strand shown runs from 5’ 3’
Later: other strand runs 3’ 5’: antiparallel |
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Linus Pauling: the alpha-helices of polypeptide chains provided the models for DNA structure
1949: Erwin Chargaff: numbers of A’s = T’s; G’s = C’s; purines = pyrimidines
1953: James Watson and Francis Crick: DNA is a complementary double helix |
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| DNA as Template for it’s Own Replication: DNA DNA |
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From Chargaff: Sequence of bases in the two chains of any given double helix have a complementary relationship.
Therefore, the sequence of any DNA strand exactly defines that of its partner
Two strands are said to be complementary in base sequence
Gene then became a real molecular object!
Excitement over the idea that if the two intertwined strands had a complementary structure, then one strand could serve as the specific surface (template) for the synthesis of the complementary strand.
Thus, this led to idea of how genes can replicate. If genes are on DNA, then one strand (containing genes) may serve to synthesize the complement (and vice-versa). |
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| Contribution of Biochemistry |
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Definition
DNA became an object of study for biochemists.
1956: Arthur Kornberg: DNA synthesis in cell free extracts of bacteria
Specific polymerizing enzyme needed: DNA polymerase I (Pol I)
Requires dATP, dCTP, dGTP, and dTTP substrates (energy rich).
Requires DNA template to determine sequence of the DNA strand that it is synthesizing
Requires Mg++
Links 3’ OH to 5’ phosphate; grows in 5’3’ direction
Strands antiparallel |
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| Each strand directs synthesis of complementary strand |
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| Enzymatic Synthesis of DNA by POL I. |
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| DNA transcription direction |
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| Direction is 5’ -> 3’ but Copied off the 3’ -> 5 ‘ strand |
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The strands of DNA are anti-parallel
The information content of the copy is not identical to that of the original template |
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Used cesium chloride (CsCl) density gradient centrifugation to demonstrate the separation of complementary strands during DNA replication.
14N and 15N differential labeling of DNA strands
Density gradient: separates Heavy, Hybrid, and Light
Heavy, DNA 15N15N, will form band at higher density (closer to bottom) in density gradient centrifugation. Light, 14N14N, will stay near top where its density matches that of salt solution. Hybrid, 15N14N, interm. |
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| DNA replication is semiconservative |
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| Semiconservative: each of two daughter DNAs should be of hybrid density (one original/one newly synthesized strand) |
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S allele of beta-globulin gene present in homozygous condition, SS, a severe anemia results. RBCs form sickle shape
+S appear normal: anemia less severe
Type of hemoglobin correlates with genetic pattern i.e. SS produces abnormal hemoglobin different in solubility from normal. +S: half normal and half abnormal ++ all normal |
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| Formation of wild-type & sickle cell hemoglobin |
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Ingram: Shows Sickle Cell hemoglobin differs from normal due to an amino acid change in beta chain
Glutamic acid replaced by valine in position 6 of beta chain.
Change in amino acid sequence observed only in patients with S allele of Beta-globin
Simplest explanation is that the S allele of the gene encodes the change in beta-globin. Gene amino acids |
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| Genes Control Amino Acid Sequences |
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Definition
Look at structure of double helix: obvious that genetic specificity has to reside in the linear sequences of their four nucleotide bases.
No information could be encoded by sugar-phosphate backbone.
4 bases (4 letter alphabet) taken three (words) at a time.
4 x 4 x 4 or 43 combinations; i.e. 64 words (Table 2-3, later)
Need to code for 20 amino acids: degeneracy
Typical gene: 1000 base pairs; # potential genes of that size is 41000; much greater than the # of genes in any organism. |
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1953: Paul Zamecnik: developed cell free extracts for protein synthesis
Radiolabeling of Amino acids
Preparative Ultracentrifugation: cell fractionation: protein synthesis pinpointed to the ribosomes.
Zamecnik and Hoagland: prior to incorporation into protein amino acids attach to Transfer RNA |
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Holley determines
Structure of t-RNA.
Anticodon recognizes
Codon on template
RNA. |
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| Amino acids attach to tRNA after reaction with ATPà amino acyl-AMP à Aminoacyl-tRNA |
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| Ribosomal RNA is not the Information Carrier |
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85% Cellular RNA found in ribosomes & amounts increased in protein synthesis
All composed of two unequally sized units
All the RNAs of small and large units are of about similar chain length.
Base composition of both units is approximately the same in all known bacteria, plants, animals.
Problems: Thus information content is small, a great extent of conservation exists across species that can not explain species diversity.
Thus rRNA cannot be information carrier. |
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| Messenger RNA is the Information Carrier during Protein Synthesis |
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Definition
Studies on Bacteriophage T4
Phage (T4) infection turns off host RNA synthesis.
Only RNA synthesized is off T4 DNA
T4 RNA has base composition similar to T4 DNA
T4 RNA does not bind to ribosome proteins
T4 RNA attaches to ribosomes and moves along their surface to bring bases in position where they can bind to appropriate tRNA-aa precursors resulting in ordering the aa. |
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| Transcription & Translation |
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Definition
Nucleotides of mRNA are assembled to form a copy of one strand of DNA.
Each group of 3 is a codon that is complementary to 3 nucleotides in anti-codon region of specific tRNA that carries the specific amino acid. RNA is the template for protein synthesis. |
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| Transcription and Translation (pic) |
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| Messenger RNA was First Overlooked as Template |
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Only small % of total cellular RNA is mRNA.
But messenger RNA shows expected large variations in length and nucleotide composition required to encode the many proteins found in a cell.
Because only a small segment of mRNA is attached at a given moment to a ribosome, a single mRNA molecule can be simultaneously read by several ribosomes. |
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| Jerard Hurwitz & Sam Weiss |
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Jerard Hurwitz & Sam Weiss: isolated the first RNA polymerases
Requires DNA template
Requires ATP, GTP, CTP, & UTP substrates
New chain growth in 5’3’ direction
Direct evidence that DNA lines up the correct ribonucleotide precursors came from seeing that RNA base composition varied with the addition of DNA molecules with different AT/GC ratios: i.e. RNA AU/GC ratio similar to DNA AT/GC ratio. |
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| Confirmation of Movement of mRNA from Nucleus to Ribosome Containing Cytoplasm by Pulse Labeling Experiment |
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Briefly expose cells to radiolabeled ribonucleotide precursors, then add large excess of unlabeled precursors.
RNA synthesized during short time window was labeled.
Showed RNA synthesized in nucleus
Within 1 hr, most of label (RNA) had left the nucleus and observed in cytoplasm |
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| Establishing the Genetic Code |
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Yanofsky & Sidney Brenner: Colinearity exists: successive nucleotides along a DNA chain code for successive amino acids along a given polypeptide chain.
1961, Brenner and Crick, groups of three nucleotide/bases are used to code individual amino acids. 20 needed; 4 x 4 x 4 = 64
1961: Nirenberg & Heinrich Matthaei showed that synthetic polynucleotide poly U (UUU….) added to cell free protein synthesis system lead to synthesis of poly Phe (Phe-Phe-…).
Khorana used polynucleotides like AGUAGU… were critical to test more specific sets of codons.
By 1966: 61/64 codons corresponded to amino acids, with most amino acids being coded for by more than one nucleotide triplet: i.e. degeneracy.
Not ambiguous |
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| Synthetic Polynucleotides |
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Definition
Poly U = Phenyl Alanine
Poly A = Lysine
Poly C = Proline
Poly AC 2 x 2 x 2 combinations
CCC CCA CAC CAA ACC ACA AAC AAA
If change proportions of A or C, certain codon base combinations in higher % ie higher C, CAC histidine CCA proline and ACC Thr |
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2 x 2 x 2 combinations
CCC CCA CAC CAA ACC ACA AAC AAA |
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| Start and Stop Signals Are Also Encoded within DNA |
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Translation of mRNA starts at one end and finishes when entire message is translated into amino acids? No
Translation both starts and stops at internal positions.
Signals must be present within DNA (& its RNA products)
Nonsense codons: UAA, UAG, & UGA: do not direct addition of an amino acid (STOP codons)
AUG initiates all polypeptide chains: pro vs eu |
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| Incorporation of Radioactively Labeled Amino Acids into Polypeptide Chains. |
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A= Distribution of radioactivity (shown in blue) among completed chains after a short period of labeling.
indicate sites of trypsin cleavage of beta globin protein.
B = Incorporation of label normalized to the length of each peptide is plotted as function of the position of the peptide within the completed chain.
Direction: NH2 COOH |
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Advent of automated DNA sequencing methods has led to determination of complete genome sequences for 100s of organisms.
Human Genome: 3 billion base pairs, contains > 20,000 genes. In chapter 20, 25,000 genes that code for proteins.
Invertebrates: 15,000 protein coding genes
Now can compare predicted amino acid sequences encoded by similar genes from different organisms.
Allows us to sometimes identify important regions of protein
Ex. Amino acids in DNA polymerases that are important for binding the incoming nucleotide are conserved in DNA pols from many different organisms.
Also, offer insights into DNA sequences that do not code for protein: Ex regulatory sequences that control expression of genes.
Comparison of genomes between individuals of same species has potential to identify mutations that lead to disease.
Rest of text: how DNA functions as template for biological complexity |
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