Chapter 14: Gene Expression: From Gene to Protein

Gene expression is the multi-step mechanism by which DNA determines the synthesis of proteins or, in some instances, specific RNA molecules that participate in protein synthesis.
This process serves as the vital link between an organism's genotype and its phenotype.
The flow of information is primarily categorized into two major stages: transcription and translation.
In eukaryotes, these stages are physically separated by the nuclear envelope, whereas in prokaryotes, such as bacteria, translation can occur simultaneously as an RNA transcript is still being synthesized because they lack a nucleus.
The central dogma of molecular biology establishes the direction of this flow: DNA acts as the template for RNA, which then serves as the template for protein production.

The connection between genes and metabolic processes was first proposed in $1902$ by Archibald Garrod, who theorized that inherited diseases resulted from a person's inability to produce specific enzymes.
Beadle and Tatum provided further evidence by studying nutritional mutants of haploid bread mold. Their experiments identified specific mutants that could not complete certain steps in the biosynthesis of the amino acid arginine.
These findings led to the development of the "one gene–one enzyme" hypothesis, which was later refined to "one gene–one protein" and eventually "one gene–one polypeptide" to account for non-enzymatic proteins and proteins consisting of multiple polypeptide chains.

The genetic code is based on a triplet code, which consists of nonoverlapping sequences of three nucleotide words.
There are $4$ nucleotide bases used to specify the $20$ standard amino acids.
Through computational combinations, there are $64$ possible codons: $61$ represent amino acids, and $3$ serve as stop signals to terminate translation.
A significant feature of the code is its redundancy—multiple codons can code for the same amino acid—but it is never ambiguous, meaning one specific codon never codes for more than one amino acid.
The code is nearly universal across all life forms, from bacteria to complex eukaryotes, suggesting it was established in a very early common ancestor.

Transcription involves the synthesis of messenger RNA (mRNA) based on a DNA template strand.
The process is catalyzed by the enzyme $\text{RNA polymerase}$ , which facilitates the separation of DNA strands and the assembly of RNA nucleotides in a $5' \text{ to } 3'$ direction.
Transcription occurs in three distinct phases:
- Initiation: $\text{RNA polymerase}$ binds to a specific DNA sequence called the promoter. In eukaryotes, this often involves a specific sequence known as the TATA box and requires the assistance of multiple transcription factors to form the transcription initiation complex.
- Elongation: The enzyme moves along the DNA, untwisting the helix and adding nucleotides at a rate of approximately $40 \text{ nucleotides per second}$ in eukaryotes. Multiple enzymes can transcribe a single gene at the same time.
- Termination: In bacteria, transcription stops at a specific terminator sequence. In eukaryotes, the enzyme transcribes a polyadenylation signal sequence, and the transcript is released approximately $10 \text{ to } 35 \text{ nucleotides}$ beyond that point.

Before leaving the nucleus, eukaryotic pre-mRNA undergoes modifications including the addition of a $5' \text{ cap}$ (a modified guanine nucleotide) and a $3' \text{ poly-A tail}$ . These alterations protect the molecule from degradation and assist in export to the cytoplasm.
RNA splicing involves the removal of non-coding regions called introns and the joining of coding regions called exons.
This process is typically mediated by spliceosomes, which are complexes of proteins and small RNAs. In some cases, ribozymes (RNA molecules acting as enzymes) can catalyze their own splicing.
Alternative RNA splicing allows a single gene to produce different polypeptides depending on which segments are treated as exons.

Translation is the process wherein a cell interprets an mRNA message and builds a polypeptide.
Facilitating this is transfer RNA (tRNA). Each tRNA has an anticodon on one end that base-pairs with a specific mRNA codon, and a corresponding amino acid on the other end.
The enzyme aminoacyl-tRNA synthetase is responsible for correctly matching a tRNA with its specific amino acid.
Ribosomes are the sites of protein synthesis, composed of a large and small subunit made of protein and ribosomal RNA (rRNA). They contain three specific sites:
- P site: Holds the tRNA carrying the growing polypeptide chain.
- A site: Holds the tRNA carrying the next amino acid to be added.
- E site: The exit site where discharged tRNAs leave.
The translation cycle consists of:
- Initiation: The small subunit binds to mRNA and an initiator tRNA (carrying methionine at the start codon $AUG$ ), followed by the attachment of the large subunit.
- Elongation: Amino acids are added one by one via codon recognition, peptide bond formation, and translocation.
- Termination: When a stop codon reaches the A site, a release factor adds a water molecule instead of an amino acid, causing the assembly to dissociate.

Following translation, polypeptides fold into their native three-dimensional shapes. Many undergo post-translational modifications, such as the addition of sugars, lipids, or phosphate groups.
Ribosomes may be free in the cytosol or bound to the endoplasmic reticulum (ER). Proteins destined for the endomembrane system or secretion are marked by a signal peptide.
The signal-recognition particle (SRP) identifies this signal peptide and directs the ribosome to a receptor on the ER membrane, where synthesis continues directly into the ER lumen.

Mutations are changes in the genetic material that can lead to altered protein structure and function.
Point mutations involve changes in a single nucleotide pair.
Substitutions include:
- Silent mutations: No change in amino acid due to code redundancy.
- Missense mutations: One amino acid is changed to another.
- Nonsense mutations: A codon is changed into a stop codon, usually resulting in a nonfunctional protein.
Insertions and deletions can cause frameshift mutations, which alter the reading frame of the entire genetic message downstream of the mutation.
A notable example is sickle-cell disease, caused by a point mutation in the $\beta \text{-globin}$ gene where a single nucleotide change results in the substitution of valine for glutamic acid.

The definition of a gene has progressed from a discrete unit of inheritance to a specific DNA sequence coding for a polypeptide.
The modern comprehensive definition describes a gene as a region of DNA that can be expressed to produce a final functional product, which may be either a polypeptide or a functional RNA molecule.