The Search for the Hereditary Molecule

By 1920, the Chromosomal Theory of Inheritance and the chemical composition of chromosomes have been revealed A. Chromosomal Theory of Inheritance revealed that genes are born on chromosomes B. Chemical composition of chromosomes: only proteins and nucleic acids, not carbohydrates or lipids C. therefore the hereditary molecule (genes) must be composed of proteins or nucleic acids

The search is on: false starts point to protein

A.P. Levene - The Tetramer Structure of DNA

Nucleotides S P B

Four kinds of DNA nucleotides

The tetramer structure of DNA hypothesis 2. Tetramer structure of DNA hypothesis:

Levene’s Problems? First (poor) techniques for measuring nucleotide composition Drew an important conclusion from tenuous data Was not suitably tentative about these conclusions

The “Alphabet Analogy” A reliance on logical argument alone: 1. the hereditary molecule must be capable of coding for vast amounts of information 2. If the hereditary molecule is a protein or NA (both polymers), then it is likely to be composed of subunits that are functionally analogous to letters of an alphabet, with the arrangement of the subunit “letters” spelling out different biological “words” (functions). 3. proteins have 20 subunits (amino acids), DNA only has 4 (nucleotides) 4. this suggested that proteins should be able to store more information...in fact, it seemed preposterous that all the complexity of life could be spelled out with an alphabet of 4 letters

Problems? The investigators underestimated the length of biological “words” (genes) Reliance on logic alone hindered demonstration that the explanation was false

The trail is found: circumstantial evidence points to DNA Avery et al. (1944) Chargaff (1949) Hershey and Chase (1952)

Griffiths & Transformation

Avery et al, 1944; the transformation bioassay is applied

Problems of Interpretation? a) bacteria are very different from all other life forms, which suggested to researchers then that bacteria might have resulted from a completely different origin of life...so they might have their own unique hereditary molecule. unwise to generalize to other organisms then b) circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Chargaff, 1949 Levene’s examination of the nucleotide composition of DNA is repeated with refined methods 1. question: what is the nucleotide composition of DNA like and does it reveal whether DNA could be the hereditary molecule? 2. methods: a) measured the nucleotide composition of DNA (repeating Levene’s work with more refined methods) b) for many species, covering the whole spectrum of life (to contend with detractors of Avery’s work who were wary about the limited utility of bacterial studies)

Chargaff, 1949 Levene’s examination of the nucleotide composition of DNA was repeated with refined chromatographic methods 3. results: a) each species has its own unique nucleotide composition, as you would expect for the hereditary molecule b) Chargaff’s Rule: [A] = [T] and [C] = [ G]...nobody understood why. This suggested that DNA had a structural similarity, and therefore a functional similarity (heredity?) in every species 4. problem of interpretation: circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Hershey & Chase, a second bioassay 1. question: is the hereditary molecule protein or DNA? 2. background: a) viruses inject their hereditary molecule into bacterial host cells, then from inside they take over the cell to reproduce b) therefore, the viruses themselves separate their hereditary molecule from the rest of their bodies (ghosts) ghosts

Hershey & Chase, a viral bioassay Protein *** DNA Protein DNA *** virus injects hereditary molecule into host cell shaker dislodges ghosts from hosts centrifuge separates ghosts from hosts virus injects hereditary molecule into host cell shaker dislodges ghosts from hosts centrifuge separates ghosts from hosts radioactivity: *** ghosts hosts ghosts hosts

Hershey & Chase, a viral bioassay problem of interpretation: circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Watson and Crick, 1953

A critical test of two perspectives: A long-standing view of life: V I T A L IS M Life is composed of special processes that cannot be studied or understood by man Understanding life as a mechanistic process: 1. Life is the product of evolution 2. Evolution is the product of reproduction 3. Reproduction is the foundation of life Could the structure of the hereditary molecule reveal the mechanistic basis of life?

The hereditary molecule’s structure was expected to reveal: The molecular basis of REPRODUCTION The molecular basis of MUTATION The molecular basis of INFO STORAGE The molecular basis by which STORED INFO IS TRNASFORMED TO BIOLOGICAL ACTIVTY

Watson and Crick’s Approach They possessed clues about DNA’s 3D shape, dimensions, and nucleotide composition They would build hypothetical models, whose dimensions could be compared to the known dimensions of DNA They wanted to examine whether a structure whose dimensions fit would explain the molecular basis of the hereditary functions …the big prize

Linus Pauling developed this method when he discovered the alpha helix secondary structure of proteins

Electron Microscopy Imaging DNA is long and thin Diameter = 20 A °

X-ray Diffraction Clues about 3D shape: several important dimensions of the molecule

Rosiland Franklin X-ray Diffraction Image of DNARosiland Franklin

Chargaff’s Rule Nucleotide composition of DNA: [A] = [T] and [C] = [G] Why?

Nucleotides are organized into polynucleotide strands with bases deployed as side chains

3D Molecular Modeling Build hypothetical structures Measure dimensions Compare with known dimensions of DNA

First Consideration: How many colinear strands? 20 A ° too small 20 A ° maybe 20 A ° maybe 20 A ° too big

First considered 3 stranded models

Two stranded models: First, tried bases outNext, tried bases in didn’t work

Two stranded models with bases facing in: Measured base pairs: purine - purine pyrimidine - pyrimidine purine - pyrimidine

Possible purine - pyrimidine pairs: A - C A - T G - C G - T Chargaff’s Rule

Only A-T and G-C pairs form hydrogen bonds (A-C and G-T cannot because their partial charges are not oriented favorably) C-GA-T

A closer look at the C-G base pair

A closer look at the A-T base pair

The hereditary molecule’s structure was expected to reveal: The molecular basis of REPRODUCTION The molecular basis of MUTATION The molecular basis of INFO STORAGE The molecular basis by which STORED INFO IS TRNASFORMED TO BIOLOGICAL ACTIVTY

The molecular basis of REPRODUCTION DNA is double stranded The two strands can UNZIP Single strands can act as TEMPLATES to order the synthesis of a second strand Addition of only COMPLEMENTARY nucleotides to a new elongating strand ensures that sequence information is preserved in new strands

REPLICATION

The molecular basis of MUTATION Accidental addition of non-complementary nucleotides to a new elongating strand or Chemical modification of a nucleotide, rendering it non- complementary

The molecular basis of INFORMATION STORAGE Neighboring nucleotides with a nucleotide pair are complementary (contributing to ability to reproduce) Neighboring nucleotides within a polynucleotide strand suffer no such limitation - any nucleotide can occupy such a site Information is stored as specific nucleotide sequences within polynucleotide strands (or sequences of nucleotide pairs)

The molecular basis of TRANSFORMATION OF STORED INFORMATION TO BIOLOGICAL ACTIVITY Required elucidation of transcription, translation, and the genetic code about 15 years

The Beginning