Source: UCF Molecular Biology I, Chapters 1–3
Tags: DNA, RNA, genetic material, transformation, Griffith, Avery, Hershey-Chase, Chargaff, central dogma, transcription, translation, codon, anticodon, tRNA, mRNA, rRNA, wobble, genetic code, ribosome, Kornberg, Ingram, sickle cell
Difficulty: Intermediate Prerequisites: Chapter 1 notes (Mendelian genetics basics). Familiarity with the general structure of cells (nucleus, cytoplasm, ribosomes).
Before molecular biology could exist as a field, scientists had to answer a deceptively simple question: what is the genetic material? This chapter traces the experiments that identified DNA (not protein) as the carrier of hereditary information, then follows the logic from DNA structure through to how proteins are made. If Chapter 1 was about inheritance patterns, this chapter is about the physical molecules behind those patterns. Everything that comes later in the course, from replication to gene regulation, builds on the central dogma introduced here.
A series of landmark experiments (Griffith, Avery, Hershey-Chase) proved DNA is the genetic material. DNA differs from RNA in its sugar (deoxyribose vs. ribose) and one base (thymine vs. uracil). The central dogma describes information flow: DNA is transcribed into RNA, which is translated into protein. The genetic code is a triplet code, it is degenerate (multiple codons per amino acid) but not ambiguous (each codon specifies only one amino acid), and tRNA serves as the adapter molecule between nucleic acid sequence and protein sequence.
Transformation (Griffith's experiment)
The process by which a non-virulent bacterium (R strain of S. pneumoniae) takes up genetic material from a dead virulent strain (heat-killed S strain) and becomes virulent itself. Griffith showed transformation occurred but did not identify DNA as the transforming agent. The R strain could not synthesise a capsule before transformation.
In simple terms, dead bacteria released something that changed live harmless bacteria into dangerous ones.
Avery, MacLeod, and McCarthy experiment
Extended Griffith's work by using enzymatic degradation to show that DNA, not protein or RNA, was the transforming material. They treated the extract with DNase, RNase, and protease separately; only DNase destroyed transforming ability.
Think of it as: they systematically eliminated suspects until DNA was the only molecule left that could explain transformation.
Hershey-Chase experiment
Used bacteriophages (viruses that infect bacteria) labelled with radioactive sulfur (^35S, in protein) and radioactive phosphorus (^32P, in DNA) to show that DNA, not protein, enters the host cell and directs phage replication. This confirmed DNA is the genetic material of bacteriophages.
In simple terms, they tracked which part of the virus actually goes inside the bacterium, and it was the DNA.
Chargaff's rules
In any double-stranded DNA molecule, the amount of adenine equals the amount of thymine (A = T), and the amount of guanine equals the amount of cytosine (G = C). Equivalently, the ratio of purines to pyrimidines is approximately 1:1.
Think of it as: the base-pairing rules written in chemistry before anyone knew the double helix structure.
Kornberg's experiment
Demonstrated the directionality of DNA synthesis: DNA is synthesised in the 5' to 3' direction. Arthur Kornberg also isolated DNA polymerase I.
Vernon Ingram and sickle cell anaemia
Showed that the sickle cell S allele of the beta-globin gene results in a single amino acid change: valine replaces glutamate at position 6. This demonstrated that DNA controls the amino acid sequence of polypeptides, and that a single base change can alter protein function.
Nucleotide
A monomer of nucleic acids, composed of a nitrogenous base, a five-carbon sugar, and one or more phosphate groups.
Nucleoside
A nucleotide without the phosphate group: just the base plus the sugar.
Purine
A double-ringed nitrogenous base. In DNA and RNA: adenine (A) and guanine (G). Think of it as: purines are "pure" and have two rings.
Pyrimidine
A single-ringed nitrogenous base. In DNA: cytosine (C) and thymine (T). In RNA: cytosine (C) and uracil (U).
Central dogma
The principle that genetic information flows from DNA to RNA to protein. DNA is transcribed into mRNA, and mRNA is translated into protein. Note: the central dogma does not say DNA goes to rRNA to protein, nor that proteins can be reverse-transcribed. None of the "trick" phrasings in the exam are correct; the proper flow is DNA → mRNA → protein.
mRNA (messenger RNA)
The RNA molecule that carries the coding sequence from DNA to the ribosome for translation. It was initially overlooked because its concentration in the cell is lower than rRNA and tRNA, leading early researchers to consider it insignificant.
tRNA (transfer RNA)
The adapter molecule between mRNA codons and amino acids. It contains an anticodon loop (which base-pairs with the mRNA codon) and an acceptor stem at the 3' end (where the amino acid attaches). tRNA contains many modified bases, such as pseudouridine and inosine.
rRNA (ribosomal RNA)
The structural and catalytic RNA component of ribosomes. It cannot be the information-carrying molecule because it is highly conserved across species and does not vary enough to account for the diversity of proteins.
Codon
A triplet of nucleotides on mRNA that specifies a particular amino acid (or a stop signal). Early molecular biologists deduced codons must be at least triplets because a two-nucleotide code would yield only 4^2 = 16 combinations, insufficient for 20 amino acids.
Genetic code degeneracy
Multiple different codons can encode the same amino acid. The code is degenerate but not ambiguous: each codon always specifies only one amino acid. You cannot easily reverse-engineer the DNA sequence from an amino acid sequence because of this degeneracy.
Wobble position
The third nucleotide position of a codon, where base-pairing with the tRNA anticodon is less strict. This allows a single tRNA to recognise multiple codons that differ only at the third position. The wobble position contributes to the degeneracy of the genetic code and means fewer distinct tRNAs are needed. Inosine in the anticodon can pair with U, C, or A at the wobble position.
Aminoacyl-tRNA synthetase
The enzyme that activates amino acids using ATP and loads (charges) them onto the correct tRNA. It does not catalyse peptide bond formation (the ribosome does that).
Shine-Dalgarno sequence
A ribosome-binding site on prokaryotic mRNA, upstream of the start codon. The small ribosomal subunit recognises this sequence to initiate translation in prokaryotes.
Kozak sequence
The equivalent translation initiation signal in eukaryotes. The small subunit of the ribosome binds the Kozak sequence (surrounding the AUG start codon) to begin translation.
Release factor
A protein that recognises a stop codon during translation and triggers release of the completed polypeptide from the ribosome.
Pulse labelling
An experimental technique in which cells are briefly exposed to radioactive precursors. Used to establish that mRNA migrates from the nucleus to the cytoplasm, and to determine the directionality of protein synthesis (N-terminus to C-terminus).
Early biochemists recognised protein synthesis was more complex than lipid or carbohydrate synthesis because proteins are non-repetitive heteropolymers (each one has a unique sequence of 20 different amino acids).
Griffith's experiment: heat-killed S strain + live R strain = live S strain recovered. The R strain took up something from the dead S cells. Griffith did not identify what the transforming substance was, and his experiment did not confirm DNA specifically.
Avery, MacLeod, and McCarthy: identified DNA as the transforming material by systematically degrading different macromolecules.
Hershey-Chase: confirmed DNA (not protein) is the genetic material using bacteriophage labelling with ^32P and ^35S.
DNA uses deoxyribose sugar; RNA uses ribose (which has a 2' OH group).
DNA uses thymine; RNA uses uracil. Both DNA and RNA have adenine, guanine, and cytosine.
Both DNA and RNA are made of covalently linked nucleotides and have a 3' OH.
A nucleotide = base + sugar + phosphate. A nucleoside = base + sugar (no phosphate).
Identifying structures: if the sugar has two OH groups on the ribose ring (2' and 3'), it is a ribonucleotide/ribonucleoside. If the base is double-ringed, it is a purine.
A = T and G = C in double-stranded DNA.
If a DNA fragment is 20% adenine, then thymine is also 20%. That accounts for 40% of bases, leaving 60% for G + C combined, so cytosine = 30%.
Chargaff showed the ratio of G to C is approximately equal to one (and A to T is approximately equal to one). He did not show A = C or G = T.
DNA → (transcription) → mRNA → (translation) → Protein.
Transcription uses the template (antisense) strand of DNA to produce mRNA. The mRNA sequence matches the gene (sense/coding) strand, except with U replacing T.
Translation reads mRNA in the 5' to 3' direction, producing protein from the N-terminus to the C-terminus.
Working out the anticodon from a DNA sequence:
Gene strand (sense): 5'-GCA-3'
Template strand: 3'-CGT-5'
mRNA (transcribed from template, same as gene strand with U for T): 5'-GCA-3'
tRNA anticodon pairs with mRNA codon in antiparallel fashion: 3'-CGU-5', which written 5' to 3' is 5'-UGC-3'. But note: the mRNA codon here is GCA, so the anticodon (antiparallel) is 5'-UGC-3'. Wait, let me reconsider. The codon is 5'-GCA-3'. The anticodon pairs antiparallel: 3'-CGU-5'. Written 5' to 3': 5'-UGC-3'. The answer is 5'-UGC-3'.
The genetic code is degenerate (redundant) but not ambiguous.
Inosine at the wobble position of the tRNA anticodon allows that tRNA to pair with multiple codons.
tRNA is the adapter molecule between mRNA and protein (not between DNA and protein directly, though the information originates from DNA).
The amino acid attaches to the 3' end of the tRNA at the acceptor stem (CCA tail), not the 5' end.
The anticodon loop base-pairs with the mRNA codon.
tRNA contains many modified bases (pseudouridine, dihydrouridine, inosine, etc.).
In prokaryotes, translation starts when the small ribosomal subunit binds the Shine-Dalgarno sequence on the mRNA.
In eukaryotes, translation starts when the small subunit binds the Kozak sequence.
The sigma factor is involved in transcription initiation (not translation). Do not confuse these.
Translation ends when a release factor binds a stop codon. The release factor is a protein, not a tRNA.
Protein synthesis proceeds from the N-terminus to the C-terminus. Pulse-labelling experiments confirmed this: radioactive amino acids appear first at the C-terminus of completed proteins (they were added most recently), while the N-terminal end, synthesised first, is "cold."
DNA replication is semi-conservative: each daughter molecule retains one parental strand and one new strand.
In a density-shift experiment starting in regular (^14N) medium and switching to heavy (^15N) medium: generation 0 = all light; generation 1 = all intermediate (one light strand, one heavy); generation 2 = half intermediate, half heavy; generation 4 = mostly heavy, small fraction intermediate.
This is the reverse of the classic Meselson-Stahl setup (which went heavy to light), but the logic is identical.
In the pulse-labelling experiment for protein synthesis directionality, chromatography separated complete proteins from incomplete fragments. This was important because incomplete fragments would contain radiolabelled amino acids at the C-terminus (the most recently added end), and mixing them with completed proteins would confuse the signal about where radioactivity concentrates in finished polypeptides.
Base composition calculation: If %A = x in double-stranded DNA, then %T = x, and %G = %C = (100 − 2x) / 2.
Anticodon determination: Gene (sense) strand → same sequence as mRNA (with U for T) → anticodon is the reverse complement of the codon, written 5' to 3'.
Sickle cell disease is one of the clearest examples of how a single nucleotide change in DNA translates to a single amino acid substitution in a protein, which then alters the protein's behaviour (haemoglobin polymerisation under low oxygen). This principle underpins all of modern genetic diagnostics. The Hershey-Chase experiment's logic (label different molecules with different isotopes to track them) remains a workhorse approach in molecular biology research.
Students often think Griffith's experiment identified DNA as the transforming agent. It did not. Griffith showed transformation occurs; Avery, MacLeod, and McCarthy identified the agent as DNA.
The central dogma does not say DNA → rRNA → protein. It says DNA → mRNA → protein. rRNA is a structural/catalytic component of the ribosome, not the information carrier.
Students confuse the wobble position as being on the tRNA. It refers to the third position of the mRNA codon, but the flexible pairing occurs at the first position of the tRNA anticodon (which pairs with codon position 3).
Aminoacyl-tRNA synthetase charges tRNAs with amino acids. It does not catalyse peptide bond formation. The ribosome (specifically, the large subunit's peptidyl transferase activity) does that.
⚠️ Know which experiment proved what. Griffith = transformation exists. Avery = DNA is the transforming agent. Hershey-Chase = DNA is the genetic material of phages. Kornberg = 5' to 3' synthesis. Chargaff = A=T, G=C.
⚠️ Be able to calculate base composition from a given percentage of one base in double-stranded DNA.
⚠️ Understand the difference between nucleotide and nucleoside, purine and pyrimidine, and ribose and deoxyribose.
⚠️ Know how to trace from a DNA sequence to the mRNA codon to the tRNA anticodon, keeping track of 5' to 3' directionality.
⚠️ Translation initiation differs between prokaryotes (Shine-Dalgarno) and eukaryotes (Kozak). This is a common exam distinction.
⚠️ The genetic code is degenerate, not ambiguous. Know what both words mean in this context.
True or false: Griffith's experiment proved that DNA is the genetic material. ___
Fill in the blank: In double-stranded DNA, if 18% of bases are guanine, then ___% are adenine.
True or false: The amino acid attaches to the 5' end of the tRNA. ___
Fill in the blank: Translation in prokaryotes initiates at the ___ sequence.
True or false: The wobble position allows one codon to specify multiple amino acids. ___
Answers: 1. False (Griffith showed transformation; Avery identified DNA). 2. 32% (G=C=18%, so A+T = 64%, A = 32%). 3. False (it attaches at the 3' acceptor stem). 4. Shine-Dalgarno. 5. False (the wobble position allows one tRNA to recognise multiple codons, not one codon to specify multiple amino acids).
Q: What did the Hershey-Chase experiment demonstrate?
A: That DNA, not protein, is the genetic material of bacteriophages. They showed this by labelling phage DNA with ^32P and phage protein with ^35S, then tracking which label entered the bacterial cell.
Q: A double-stranded DNA fragment is 20% adenine. What percentage is cytosine?
A: 30%. A = T = 20%, so A + T = 40%. The remaining 60% is G + C, split equally: C = 30%.
Q: Why was mRNA initially overlooked as the information carrier for protein synthesis?
A: Its concentrations in the cell were below those of rRNA and tRNA, so it was thought to be insignificant.
Q: What is the function of aminoacyl-tRNA synthetase?
A: It activates amino acids using ATP and loads them onto the correct tRNA. It ensures the right amino acid is paired with the right tRNA (the "second genetic code").
Q: Given gene strand 5'-GCA-3' and template strand 3'-CGT-5', what is the tRNA anticodon?
A: 5'-UGC-3'. The mRNA codon (same as gene strand, with U for T) is 5'-GCA-3'. The anticodon pairs antiparallel: 3'-CGU-5', which written 5' to 3' is UGC.
The experiments in this chapter (Griffith, Avery, Hershey-Chase) establish the molecular identity of genes, which directly feeds into Chapter 3's discussion of chemical bonds and thermodynamics (how DNA holds together, how proteins fold). The central dogma framework introduced here is the scaffold for every later topic: replication (DNA → DNA), transcription regulation, RNA processing, and translation control. Sickle cell anaemia reappears in discussions of point mutations and protein structure.
DNA, RNA, genetic material, transformation, Griffith experiment, Avery MacLeod McCarthy, Hershey-Chase, bacteriophage, Chargaff's rules, base composition, purine, pyrimidine, nucleotide, nucleoside, deoxyribose, ribose, thymine, uracil, central dogma, transcription, translation, mRNA, tRNA, rRNA, codon, anticodon, wobble position, inosine, degeneracy, genetic code, aminoacyl-tRNA synthetase, Shine-Dalgarno, Kozak sequence, release factor, pulse labelling, Kornberg, Vernon Ingram, sickle cell, beta-globin, semi-conservative replication, Meselson-Stahl, protein synthesis directionality, N-terminus, C-terminus