Difficulty: Intermediate | Prerequisites: Parts 1 and 2 of these notes (DNA structure, base pairing, replication)
With DNA replication established (DNA → DNA), the next problem was: how does DNA's information get used to build proteins? DNA lives in the nucleus, but proteins are made in the cytoplasm. Something has to carry the message between the two. This section covers the discovery of RNA's role as that intermediary, the three major RNA types (mRNA, tRNA, rRNA), the mechanics of transcription, and how Francis Crick's central dogma (DNA → RNA → protein) was confirmed. If you are coming in cold, the key idea is that DNA is not directly read by the protein-making machinery; instead, a temporary RNA copy is made and sent to the ribosomes.
Crick proposed the central dogma: DNA → RNA → protein. Three types of RNA carry out different jobs: mRNA carries the genetic message, tRNA delivers amino acids, and rRNA forms the structural core of the ribosome. Transcription is carried out by RNA polymerase, which reads the template strand 3' to 5' and builds mRNA 5' to 3'. Sigma factors direct initiation at promoters; rho helicase drives termination.
Central dogma of molecular biology
Crick's principle (1956) that information flows DNA → RNA → protein. The only common exception is reverse transcriptase (RNA → DNA). In simple terms, DNA is the master copy, RNA is the working copy, and protein is the final product.
Messenger RNA (mRNA)
The RNA molecule that carries a copy of a gene's sequence from DNA to the ribosome for translation. It is only 1 to 5% of total cellular RNA, which is why it was initially overlooked. Think of it as the temporary blueprint sent from the archive (nucleus) to the factory floor (ribosome).
Transfer RNA (tRNA)
A small RNA molecule (the "adaptor") that carries a specific amino acid on its 3' end and has an anticodon loop that recognises a complementary codon on mRNA. Crick predicted its existence before it was discovered. Think of it as a delivery truck with an address label (anticodon) and a cargo bay (amino acid attachment site).
Ribosomal RNA (rRNA)
The structural and catalytic RNA component of ribosomes. It makes up about 85% of total cellular RNA. It is highly conserved across species, which is precisely why it cannot be the information carrier: its sequence is too similar between organisms to account for genetic diversity.
RNA polymerase
The enzyme that synthesises RNA from a DNA template, using ATP, GTP, CTP, and UTP as substrates. Synthesis is 5' to 3'.
Sigma factor (σ)
A subunit of bacterial RNA polymerase that recognises promoter sequences and directs the enzyme to the correct start site for transcription. Different sigma factors are used under different conditions (e.g. stress, starvation).
Promoter
The DNA sequence upstream of a gene where RNA polymerase binds to initiate transcription. In bacteria, consensus sequences at positions -10 and -35 are recognised by the sigma factor.
Aminoacyl-tRNA synthetase
The enzyme that attaches the correct amino acid to its corresponding tRNA (called "charging"). There are 20, one for each amino acid. Error rate is about 1 in 10,000 (1 in 3,000 for isoleucine, which is easily confused with valine).
Anticodon
A three-nucleotide sequence on tRNA that is complementary to a codon on mRNA. It ensures the correct amino acid is delivered during translation.
In eukaryotes, DNA is confined to the nucleus, but protein synthesis occurs in the cytoplasm on ribosomes.
A second information-carrying molecule is needed to shuttle the message.
Torbjorn Caspersson and Jean Brachet observed that RNA is concentrated in the cytoplasm where protein synthesis takes place, making it a strong candidate.
Vernon Ingram (1954) showed that sickle cell haemoglobin differs from normal haemoglobin by a single amino acid substitution: glutamate → valine at position 6 of β-globin.
This was demonstrated by protein fingerprinting: trypsin digestion of haemoglobin followed by two-dimensional chromatography.
The finding proved that a change in a gene (DNA) produces a specific change in a protein's amino acid sequence, confirming the gene-to-protein link.
Homozygous S allele causes severe sickle cell anaemia; heterozygous causes milder symptoms but confers some protection against malaria (explaining higher carrier frequencies in malaria-endemic regions).
Single-stranded, four nucleotides linked by 3' to 5' phosphodiester bonds.
Two differences from DNA:
Ribose sugar (OH on the 2' carbon) instead of deoxyribose
Uracil replaces thymine (uracil lacks the methyl group)
Neither difference prevents base pairing with DNA, so RNA can be templated directly from a DNA strand.
Proposed by Crick in 1956: DNA → RNA → protein.
Two arrows are normally unidirectional.
Exceptions: reverse transcriptase (retroviruses) and telomerase can synthesise DNA from an RNA template (RNA → DNA).
An early idea was that RNA folds into cavities that physically accommodate specific amino acids.
Problem: RNA's bases interact mainly with water-soluble groups, but many amino acid side chains (leucine, valine, phenylalanine) are hydrophobic. Even if hydrophobic pockets could form, discriminating between chemically similar amino acids (alanine vs. glycine, valine vs. isoleucine) by shape alone seemed implausible.
Crick proposed instead that adaptor molecules (later identified as tRNA) first bind amino acids and then recognise the RNA template via hydrogen bonding (codon-anticodon pairing).
Paul Zamecnik (1953) created a cell-free protein synthesis system from rat liver. Showed amino acids must be "activated" by ATP before incorporation into polypeptides.
By 1956, radiolabelling and ultracentrifugation localised protein synthesis to ribosomes and showed amino acids attach to a small RNA before being incorporated. This RNA was tRNA, Crick's predicted adaptor.
Robert Holley (1964) determined tRNA's structure using specific ribonucleases and ion-exchange chromatography. Key features:
Anticodon loop: recognises the codon on mRNA
3' end (acceptor stem): where the amino acid attaches
Discriminator base: helps select the correct amino acid
Several modified bases: pseudouridine (Ψ), ribothymidine (T), dihydrouridine (DHU), inosine (I, enables wobble pairing), methylated bases
Humans have 600+ tRNA genes, though the reason for this redundancy is not fully understood.
The reaction occurs in two steps:
Amino acid + ATP → aminoacyl-AMP + PPi
Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
20 synthetases, one per amino acid. Each recognises both the acceptor stem and the anticodon loop of its target tRNA(s).
For amino acids like serine, which have six very different codons (AGC, AGU, UCA, UCU, UCC, UCG), the synthetase recognises additional structural features of the tRNA beyond just the anticodon.
Contains an editing/proofreading pocket to catch errors.
85% of cellular RNA is rRNA (~15,000 ribosomes per E. coli cell).
Prokaryotic ribosomes: 70S total (50S large subunit + 30S small subunit).
Eukaryotic ribosomes: 80S total (60S large subunit + 40S small subunit).
rRNA base composition is nearly identical across bacteria, plants, and animals, with higher GC content regardless of the organism's overall DNA AT/GC ratio.
This conservation makes rRNA useless as a genetic information carrier but excellent for bacterial identification (16S rRNA sequencing).
Initially, rRNA was assumed to be the template because of its abundance at protein synthesis sites.
In 1953, Hershey noticed that new RNA appeared after phage infection but degraded quickly, so it was dismissed.
In 1960, Brenner, Jacob, and Meselson proved mRNA's existence using phage T2:
Grew bacteria in ¹³C¹⁵N medium to make "heavy" ribosomes.
Infected with phage, transferred to medium containing ³²P.
Centrifuged: all ribosomes were old and heavy (no new ribosomes made); new ³²P-labelled RNA associated with old ribosomes.
Since phage infection shuts down host RNA synthesis, the new RNA must be phage-encoded mRNA attaching to pre-existing ribosomes.
Confirmed by hybridising the new RNA back to phage DNA.
Jerard Hurwitz and Sam Weiss isolated RNA polymerase.
Requirements: DNA template, ATP, GTP, CTP, UTP.
RNA is synthesised 5' to 3' off the 3' to 5' template strand.
The AU/GC ratio of the RNA product matches the AT/GC ratio of the DNA template, confirming faithful copying.
RNA polymerase must find the correct start site via the promoter.
In bacteria, the sigma factor recognises consensus sequences at -10 and -35 relative to the start site.
The sigma factor's helix-turn-helix motif fits into the major groove of the DNA.
Different sigma factors are deployed under different conditions (heat shock, nitrogen starvation, chemotaxis, etc.).
Rho helicase attaches to the mRNA transcript and moves along it toward the RNA polymerase.
When rho catches up, it catalyses dissociation of mRNA from both the DNA template and the polymerase.
Demonstrated by pulse-labelling experiments in eukaryotic cells.
Cells were briefly exposed to ³H-labelled cytidine. After 15 minutes, labelled RNA was found mostly in the nucleus. After 88 minutes, it had moved to the cytoplasm.
This confirmed the expected trafficking: transcription in the nucleus, translation in the cytoplasm.
Transcription direction: 5' → 3' (template read 3' → 5')
tRNA charging: Amino acid + ATP → aminoacyl-AMP + PPi; then aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
Synthetase error rate: ~1/10,000 (1/3,000 for isoleucine)
Prokaryotic ribosomes: 70S (50S + 30S); eukaryotic: 80S (60S + 40S)
mRNA is 1 to 5% of total cellular RNA
Humans have 600+ tRNA genes, 22 mitochondrial tRNA genes
The central dogma is the operating logic behind mRNA vaccines: synthetic mRNA encoding a viral protein is delivered to cells, where ribosomes translate it into protein, triggering an immune response. No DNA is altered. The entire approach depends on the same transcription-to-translation pipeline described here. Separately, 16S rRNA sequencing (possible because rRNA is so conserved) is the standard method for identifying unknown bacteria in clinical microbiology and environmental science.
Students often confuse the template strand (3' to 5', read by RNA polymerase) with the coding strand (5' to 3', same sequence as the mRNA except T instead of U). Transcription reads the template strand, not the coding strand.
rRNA is the most abundant RNA, but it is not the information carrier. Its abundance is because ribosomes are structural machines needed in large numbers, not because they store genetic messages.
tRNA does not "read" the amino acid. The aminoacyl-tRNA synthetase recognises both the tRNA and the amino acid. The tRNA's anticodon reads the mRNA codon, not the amino acid itself.
The central dogma does not say information never flows from RNA to DNA. Reverse transcriptase is a well-known exception. The dogma's core claim is that information does not flow from protein back to nucleic acid.
⚠️ Know the three RNA types, their functions, and their approximate proportions of total cellular RNA.
⚠️ Be able to write out the tRNA charging reaction in two steps.
⚠️ Understand why rRNA was initially suspected as the template and why that turned out to be wrong (conservation, not variability).
⚠️ The Brenner-Jacob-Meselson experiment for mRNA discovery is a common exam question. Know the heavy ribosome / ³²P-labelled RNA logic.
⚠️ Sigma factor, promoter consensus sequences (-10, -35), and rho-dependent termination are frequently tested.
True or False: mRNA makes up about 85% of total cellular RNA.
Fill in the blank: The sigma factor recognises ______ sequences at positions -10 and -35 on bacterial DNA.
True or False: Aminoacyl-tRNA synthetases only recognise the anticodon loop to select the correct tRNA.
Fill in the blank: Prokaryotic ribosomes sediment at ______S, composed of a 50S and a 30S subunit.
True or False: The central dogma states that information can never flow from RNA to DNA.
Answers: 1. False (rRNA is ~85%; mRNA is only 1 to 5%). 2. Promoter consensus. 3. False (they recognise both the acceptor stem and the anticodon loop, plus additional features in some cases). 4. 70. 5. False (reverse transcriptase is a well-known exception).
Q: Why was rRNA initially thought to be the information-carrying RNA, and what evidence disproved this?
A: rRNA makes up about 85% of cellular RNA and is found at sites of protein synthesis, which made it the obvious candidate. However, rRNA's base composition is nearly identical across all known organisms despite wide variation in genomic AT/GC ratios. This conservation means rRNA cannot account for genetic diversity between species. The true information carrier, mRNA, was later identified as a small, rapidly turning-over fraction of total RNA.
Q: Describe the two-step reaction by which an amino acid is attached to its tRNA.
A: First, the amino acid reacts with ATP to form aminoacyl-AMP, releasing pyrophosphate (PPi). Second, the aminoacyl group is transferred from AMP to the 3' end of the appropriate tRNA, releasing AMP. Both steps are catalysed by the same aminoacyl-tRNA synthetase.
Q: In the Brenner-Jacob-Meselson experiment, how did the researchers demonstrate that phage-encoded mRNA associates with pre-existing ribosomes rather than new ones?
A: Bacteria were grown in ¹³C¹⁵N medium, making all ribosomes "heavy." After phage infection, cells were transferred to medium with ³²P. Upon centrifugation, all ribosomes were heavy (no new light ribosomes had been made), and the new ³²P-labelled RNA was found associated with these old heavy ribosomes. Since phage infection shuts down host RNA synthesis, the new RNA must be phage-encoded mRNA using existing ribosomes for translation.
Q: Why did Crick reject the idea that RNA directly binds amino acids through shaped cavities?
A: RNA bases primarily interact with water-soluble groups, but many amino acid side chains are hydrophobic. Even if RNA could form some hydrophobic pockets, it seemed unable to discriminate between chemically similar amino acids (e.g. alanine vs. glycine, which differ by a single methyl group). This led Crick to propose adaptor molecules (tRNA) that would recognise both the amino acid and the RNA template separately.
This section completes the "DNA → RNA" portion of the central dogma, connecting directly to Part 2 (DNA → DNA, replication). Part 4 of these notes covers the final arrow: RNA → protein (translation and the genetic code). The sickle cell haemoglobin example bridges genetics and biochemistry, and reappears in courses on haematology, population genetics (malaria selection), and protein structure. The concept of sigma factors and promoters connects forward to gene regulation topics later in most molecular biology courses.
central dogma, mRNA, tRNA, rRNA, transcription, RNA polymerase, sigma factor, promoter, rho termination, aminoacyl-tRNA synthetase, tRNA charging, anticodon, codon, Crick adaptor hypothesis, Brenner Jacob Meselson, messenger RNA discovery, sickle cell haemoglobin, Vernon Ingram, pulse labelling, ribosome subunits 70S 80S, Robert Holley, Paul Zamecnik, wobble base pairing, inosine, chapter 2 molecular biology