Source: Molecular Biology I, University of Central Florida, Chapter 1
Tags: DNA replication, DNA polymerase, phosphodiester bond, RNA, rRNA, tRNA, mRNA, ribosomes, transcription, translation, central dogma, protein synthesis, Francis Crick, George Palade, BIOL 101
Difficulty: Introductory to Intermediate Prerequisites: Part 1 of these notes (DNA structure, base pairing, Chargaff's Rule). You should be comfortable with 5' and 3' directionality before reading this.
Once you understand what DNA looks like, the next question is: what does it do? This section covers the molecular machinery that copies DNA (replication), reads it into RNA (transcription), and converts that RNA message into protein (translation). Together, these processes form the Central Dogma of molecular biology. You will also meet the three major types of RNA and the ribosome, the cellular machine where proteins are assembled. This material is the engine room of the course.
DNA polymerase copies DNA in the 5' to 3' direction by forming phosphodiester bonds. The genetic information flows from DNA to mRNA (transcription) to protein (translation), a principle known as the Central Dogma. Ribosomes, built from rRNA and protein, are the sites where translation occurs. Three types of RNA, each made by a different RNA polymerase, carry out distinct roles in this process.
DNA polymerase
The enzyme responsible for DNA replication. It joins individual nucleotides (dNTPs) to a growing DNA strand by catalysing phosphodiester bond formation, always working in the 5' to 3' direction. Think of it as the molecular photocopier that reads the template and builds a new matching strand.
Phosphodiester bond
The covalent bond that links one nucleotide to the next in a DNA or RNA strand, formed between the 3'-OH of the existing strand and the alpha-phosphate of the incoming dNTP.
Deprotonation of the 3'-OH group
The 3'-OH on the sugar of the growing strand loses a hydrogen atom (proton), becoming negatively charged. This makes it highly reactive, enabling it to attack the incoming nucleotide's phosphate and form the new bond. In simple terms, the end of the strand becomes "charged up" so it can grab the next nucleotide.
dNTP (deoxyribonucleoside triphosphate)
The building blocks used by DNA polymerase during replication. Each dNTP carries three phosphate groups; two are released as pyrophosphate when the bond forms, providing the energy for the reaction.
Ribosome
The molecular machine where protein synthesis occurs. Composed of RNA (about 60%) and protein, organised into a large and a small subunit. Contains two functional sites: the decoding centre and the peptidyl transferase centre. Think of it as the factory floor where mRNA instructions are read and proteins are assembled.
rRNA (ribosomal RNA)
The RNA component of ribosomes. Makes up 60% of ribosomal mass. Cellular RNA is roughly 80% rRNA, making it the most abundant RNA class by mass.
tRNA (transfer RNA)
The "adaptor" molecule that carries specific amino acids to the ribosome during translation. Each tRNA has an anticodon that pairs with a complementary codon on mRNA. Discovered in 1957 by Paul Zamecnik. Transcribed by RNA polymerase III. The most abundant RNA by molar concentration, with tens of millions of transcripts per cell. In simple terms, tRNA is the delivery truck that brings the right amino acid to match each three-letter code on the mRNA.
mRNA (messenger RNA)
The RNA molecule that carries a copy of the genetic instructions from DNA to the ribosome. Transcribed by RNA polymerase II. mRNA abundance varies enormously between different genes and cell types.
Transcription
The process of copying a gene's DNA sequence into mRNA. DNA helicase separates the two strands, and RNA polymerase reads the template strand in the 3' to 5' direction while synthesising the mRNA in the 5' to 3' direction.
Translation
The process by which ribosomes read mRNA codons and, with the help of tRNA anticodons, assemble amino acids into a polypeptide chain (protein).
Central Dogma
Proposed by Francis Crick in 1970. The principle that genetic information flows: DNA to RNA to Protein. This is the foundational framework of molecular biology.
Protein denaturing
The unfolding of a protein's three-dimensional structure, which is generally irreversible. Notable exceptions include ribonuclease and Thermus aquaticus proteins.
George Palade
Discovered ribosomes and pioneered the study of the endoplasmic reticulum and its role in protein synthesis.
Ribosomopathies
Rare genetic disorders caused by problems in the processing, folding, or assembly of rRNA and ribosomal subunits.
DNA polymerase synthesises new DNA in the 5' to 3' direction only
The enzyme positions the 3'-OH of the existing strand (or RNA primer) next to the alpha-phosphate of the incoming dNTP
The 3'-OH is deprotonated, making it negatively charged and reactive
This charged oxygen performs a nucleophilic attack on the alpha-phosphate, forming a phosphodiester bond and releasing pyrophosphate
Each new nucleotide is added to the 3' end of the growing strand
rRNA (ribosomal RNA): structural and catalytic component of ribosomes. 80% of all cellular RNA is rRNA. Transcribed by RNA polymerase I
Prokaryotic rRNA: 5S, 16S, and 23S
Eukaryotic cytoplasmic rRNA: 5S, 5.8S, 17S/18S, and 26S/28S. The 5.8S rRNA corresponds to the 5' terminal 160 nucleotides of prokaryotic 23S rRNA
tRNA (transfer RNA): the adaptor molecule. Encoded by 619 genes across 57 anticodon families (432 unique transcripts in humans). Copy numbers are unequal. Transcribed by RNA polymerase III. Most abundant by molar concentration
mRNA (messenger RNA): carries genetic instructions from DNA to ribosomes. Abundance varies hugely between transcripts. Transcribed by RNA polymerase II
Composed of protein and RNA, organised into a small and a large subunit
RNA makes up approximately 60% of ribosomal mass
Two critical functional sites:
Decoding centre: where the mRNA codon is matched to the tRNA anticodon
Peptidyl transferase centre: where peptide bonds are formed between amino acids
Human rRNA genes are duplicated and clustered on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22
Ribosomopathies are rare genetic disorders arising from defects in rRNA processing, folding, or ribosomal subunit assembly
DNA helicase breaks the hydrogen bonds between the two DNA strands
One strand serves as the template
RNA polymerase moves along the template strand and adds complementary RNA nucleotides in the 5' to 3' direction
The product is a single-stranded mRNA molecule
Ribosomes read mRNA three nucleotides at a time (codons)
Each codon is matched by a tRNA carrying the corresponding anticodon and a specific amino acid
The ribosome links amino acids together in the order specified by the mRNA sequence, forming a polypeptide chain
The amino acid sequence of the final protein is entirely determined by the mRNA sequence
Proposed by Francis Crick in 1970
Information flow: DNA → RNA → Protein
This is a one-directional framework for how genetic information is expressed (with known exceptions like reverse transcriptase, covered later in the course)
Protein unfolding is generally irreversible once a protein loses its three-dimensional structure
Exceptions exist: ribonuclease can refold spontaneously, and proteins from Thermus aquaticus are unusually heat-stable
Direction of synthesis: All nucleic acid polymerases work 5' → 3'.
Phosphodiester bond formation: 3'-OH (deprotonated) + alpha-phosphate of incoming dNTP → new phosphodiester bond + pyrophosphate released
Central Dogma: DNA → (transcription) → RNA → (translation) → Protein
RNA polymerase assignments:
RNA Pol I → rRNA
RNA Pol II → mRNA
RNA Pol III → tRNA
Ribosomopathies, though rare, include conditions like Diamond-Blackfan anaemia. Understanding ribosome function is also central to how antibiotics work: many antibiotics (e.g. tetracycline, erythromycin) target bacterial ribosomes specifically, exploiting the structural differences between prokaryotic and eukaryotic ribosomes.
Students often think DNA polymerase can synthesise in both directions. It cannot. Synthesis is always 5' to 3'. The leading/lagging strand distinction (covered later) exists precisely because of this constraint.
"Central Dogma" does not mean information can never flow backwards. Reverse transcriptase (found in retroviruses) copies RNA back into DNA. The dogma describes the default direction, not an absolute rule.
Students sometimes confuse which RNA polymerase makes which RNA. A useful mnemonic: the numbers go in reverse order of the RNA's abundance by mass. Pol I makes rRNA (most abundant by mass), Pol II makes mRNA, Pol III makes tRNA.
Protein denaturing is not the same as protein degradation. Denaturing is loss of shape; degradation is breaking the protein into pieces.
⚠️ The direction of DNA synthesis (5' to 3') is tested constantly. Every replication and transcription question assumes you know this.
⚠️ Know which RNA polymerase transcribes which RNA type (Pol I → rRNA, Pol II → mRNA, Pol III → tRNA).
⚠️ Understand the mechanism of phosphodiester bond formation: deprotonation of 3'-OH, nucleophilic attack on alpha-phosphate.
⚠️ The Central Dogma (DNA → RNA → Protein) and its attribution to Francis Crick (1970) are standard exam material.
⚠️ Ribosome composition (60% RNA, 40% protein; two subunits) and functional sites (decoding centre, peptidyl transferase centre) are commonly tested.
⚠️ Know that rRNA genes cluster on chromosomes 13, 14, 15, 21, and 22.
True or False: DNA polymerase synthesises new DNA in the 3' to 5' direction.
Fill in the blank: RNA polymerase ___ transcribes mRNA.
True or False: tRNA is the most abundant RNA in the cell by mass.
Fill in the blank: The two functional sites of the ribosome are the ___ and the ___.
True or False: Protein denaturing is always irreversible with no exceptions.
Answers: 1. False (5' to 3'). 2. II. 3. False (rRNA is most abundant by mass; tRNA is most abundant by molar concentration). 4. Decoding centre; peptidyl transferase centre. 5. False (ribonuclease and Thermus aquaticus proteins are exceptions).
Q: Describe the chemical mechanism by which a new phosphodiester bond is formed during DNA replication.
A: The 3'-OH group on the terminal sugar of the growing strand is deprotonated, becoming negatively charged. This reactive oxygen performs a nucleophilic attack on the alpha-phosphate of the incoming dNTP, forming a new phosphodiester bond and releasing pyrophosphate.
Q: What is the Central Dogma of molecular biology, and who proposed it?
A: The Central Dogma, proposed by Francis Crick in 1970, states that genetic information flows from DNA to RNA (via transcription) to protein (via translation).
Q: Compare and contrast the three major types of RNA in terms of function, abundance, and which RNA polymerase transcribes each.
A: rRNA is the structural/catalytic RNA of ribosomes, the most abundant by mass, transcribed by Pol I. tRNA is the adaptor that delivers amino acids, the most abundant by molar concentration, transcribed by Pol III. mRNA carries genetic instructions to the ribosome, varies widely in abundance, and is transcribed by Pol II.
Q: Why are prokaryotic and eukaryotic rRNA subunit sizes different, and what are they?
A: Prokaryotic rRNA includes 5S, 16S, and 23S subunits. Eukaryotic cytoplasmic rRNA includes 5S, 5.8S, 18S, and 28S (with the 5.8S corresponding to the 5' end of prokaryotic 23S). The size differences reflect evolutionary divergence and are exploited by antibiotics that target prokaryotic ribosomes selectively.
Q: What are ribosomopathies?
A: Ribosomopathies are rare genetic disorders caused by defects in the processing, folding, or assembly of rRNA and ribosomal subunits. They result in impaired ribosome function and are associated with various developmental and haematological conditions.
DNA replication mechanics connect forward to the chapter on replication forks, Okazaki fragments, and the leading/lagging strand problem (all consequences of 5' to 3' synthesis). The three RNA types and their polymerases are foundational for the gene expression and regulation chapters. The Central Dogma is the conceptual spine of the entire course; exceptions to it (reverse transcription, RNA editing) will be covered in later chapters.
DNA replication, DNA polymerase, phosphodiester bond, nucleophilic attack, 3'-OH, dNTP, 5' to 3' synthesis, rRNA, tRNA, mRNA, ribosome, ribosomal subunits, decoding centre, peptidyl transferase centre, RNA polymerase I, RNA polymerase II, RNA polymerase III, transcription, translation, Central Dogma, Francis Crick, George Palade, protein synthesis, protein denaturing, ribosomopathies, prokaryotic rRNA, eukaryotic rRNA, adaptor molecule, Paul Zamecnik, BIOL 101