Gene Expression: From DNA to Protein

Gene Expression

Central Dogma of Molecular Biology

The central dogma of molecular biology describes the unidirectional flow of genetic information from DNA to RNA to protein. This process is fundamental to how genotype is expressed as phenotype in living organisms.

• Transcription: The process by which RNA is synthesized using DNA as a template.

• Translation: The process by which proteins are synthesized using the encoded messages of mRNA.

• Gene Expression: Sometimes refers collectively to both transcription and translation.

• DNA can be replicated, and RNA can be reverse-transcribed into DNA, but the transfer of nucleic acid information to protein is irreversible.

Example: The central dogma is summarized as DNA → RNA → Protein, with mRNA as the intermediate molecule.

Introduction to Transcription

Transcription is the process that builds RNA using DNA within a gene as the coding template. Genes are small units of DNA that encode a product, such as a protein.

• Promoter: DNA sequence where transcription begins; site of RNA polymerase attachment.

• RNA Polymerase: Enzyme that synthesizes RNA from scratch (no primer needed).

• Terminator: DNA sequence where transcription ends.

• Upstream: DNA sequences in the opposite direction of transcription.

• Downstream: DNA sequences in the direction of transcription.

Example: Promoter and terminator regions define the boundaries of transcription for a gene.

Overview of Transcription

DNA in a gene consists of two strands: the coding strand and the template strand. The RNA sequence produced during transcription is complementary to the template strand and matches the coding strand (except uracil replaces thymine).

• RNA is synthesized from the 5' to 3' end by pairing free RNA nucleotides on the DNA template.

• Base pairing follows Watson & Crick rules: A-U (in RNA), T-A, C-G, G-C.

Example: Determining the mRNA transcript from a given DNA coding strand.

Steps of Transcription

Initiation of Transcription

Transcription begins when RNA polymerase binds to the promoter and unwinds the DNA strands. In prokaryotes, RNA polymerase binds directly, while in eukaryotes, transcription factors are required.

• Unwinding exposes the template strand for RNA synthesis.

Elongation of Transcription

During elongation, RNA polymerase synthesizes the RNA molecule by pairing free RNA nucleotides with the DNA template. The enzyme moves along the DNA, unwinding it and building RNA in the 5’ to 3’ direction.

• Multiple RNA polymerases can transcribe a gene simultaneously.

Termination of Transcription

Termination occurs when RNA polymerase reaches the terminator sequence, resulting in the release of the newly synthesized RNA molecule. Prokaryotes and eukaryotes differ in their termination mechanisms; eukaryotic termination produces a pre-mRNA that requires further modification.

Eukaryotic RNA Processing & Splicing

RNA Processing

Unlike prokaryotic mRNA, eukaryotic mRNA requires modification after transcription. The initial transcript, called pre-mRNA, undergoes processing to become mature mRNA.

• 5’ Cap: Addition of a modified guanine nucleotide to the 5’ end.

• Poly-A Tail: Addition of a sequence of adenine nucleotides to the 3’ end.

• Functions: Facilitate export from nucleus, protect from degradation, and help ribosome attachment for translation.

RNA Splicing

Eukaryotic genes contain introns (noncoding regions) and exons (coding regions). RNA splicing removes introns and joins exons to produce mature mRNA. The spliceosome is the complex responsible for splicing.

• Alternative Splicing: Genes can be spliced in different ways to produce multiple products.

Types of RNA

Messenger RNA (mRNA), Ribosomal RNA (rRNA), Transfer RNA (tRNA)

Cells use several types of RNA, each with distinct functions:

• mRNA: Carries genetic information from DNA and is translated into protein. Contains codons (three-nucleotide sequences coding for amino acids).

• rRNA: Forms part of the ribosome structure.

• tRNA: Transfers amino acids to the ribosome during translation. Contains anticodons complementary to mRNA codons.

The Genetic Code

Codons and Amino Acids

The genetic code is a set of rules by which DNA/RNA sequences are translated into amino acid sequences in proteins. Each codon (three nucleotides) specifies one amino acid. The code is nearly universal and redundant (multiple codons can code for the same amino acid).

• To use the genetic code: Convert DNA coding sequence to mRNA (replace T with U), identify codon frames, and match each codon to its amino acid until a stop codon is reached.

Translation: Protein Synthesis

Introduction to Translation

Translation is the process of synthesizing proteins using the encoded messages of mRNA. Ribosomes facilitate this process by joining amino acids together.

• Ribosomes: Complexes of proteins and rRNA that build polypeptides.

• tRNA: Carries amino acids and contains anticodons that pair with mRNA codons.

• Charged tRNA: tRNA attached to an amino acid.

• Discharged tRNA: tRNA not attached to an amino acid.

Ribosome Structure and tRNA Binding Sites

Ribosomes consist of small and large subunits. Prokaryotic ribosomes are 70S (50S + 30S), while eukaryotic ribosomes are 80S (60S + 40S). Each ribosome has three tRNA binding sites:

• A-site (Aminoacyl-tRNA): Holds tRNA with the next amino acid.

• P-site (Peptidyl-tRNA): Holds tRNA with the growing polypeptide chain.

• E-site (Exit): Discharged tRNAs leave the ribosome.

Steps of Translation

Initiation of Translation

Translation begins when the small ribosomal subunit binds to mRNA and a tRNA, followed by the large subunit. The start codon (AUG) specifies methionine as the first amino acid. Initiation factors and energy are required.

Elongation of Translation

During elongation, amino acids are added one by one to the C-terminus of the growing polypeptide chain. The ribosome reads mRNA codons 5’ to 3’, pairing them with tRNA anticodons. New tRNAs enter the A-site, shift to the P-site, and exit via the E-site.

Termination of Translation

Termination occurs when a stop codon reaches the ribosome’s A-site, triggering release factors to bind. The polypeptide chain is cleaved and released, and the translation assembly disassembles.

Post-Translational Modification

Types of Modifications

After translation, proteins may undergo post-translational modifications (PTM) that regulate their activity. Common types include:

• Methylation

• Acetylation

• Ubiquitination

• Phosphorylation

• Glycosylation: Addition of carbohydrates to proteins.

Transcription vs. Translation: Comparison

Feature	Transcription	Translation
Product Formed	RNA Molecule	Protein
Macromolecule Change?	Nucleic Acid → Nucleic Acid	Nucleic Acid → Protein
Major Enzyme/Structure	RNA Polymerase	Ribosome
Location	Nucleus (Eukaryotes)	Cytoplasm
Direction of Synthesis	5' → 3'	N-terminus → C-terminus

Mutations

Types and Effects

Mutations are permanent changes in the DNA sequence. They can affect gene expression and protein function, and may be harmful, beneficial, or neutral. Mutations can occur spontaneously or be induced by environmental factors (mutagens).

• Frameshift Mutation: Insertion or deletion of nucleotides that alters the reading frame.

• Missense Mutation: Substitution that changes one amino acid.

• Nonsense Mutation: Substitution that creates a stop codon, terminating translation prematurely.

• Silent Mutation: Substitution that does not change the amino acid sequence due to redundancy in the genetic code.

Summary

Gene expression involves the transcription of DNA into RNA and the translation of RNA into protein. Eukaryotic RNA undergoes processing and splicing before translation. The genetic code determines how nucleotide sequences are converted into amino acids. Mutations in DNA can impact gene expression and protein function, contributing to genetic diversity.