Gene Expression, Transcription, and Translation: Molecular Basis of Protein Synthesis

Gene Expression and the Central Dogma

Overview of Gene Expression

Gene expression is the process by which the information encoded in DNA is used to produce proteins, primarily through the intermediary molecule RNA. This process is fundamental to cell function and phenotype determination.

• Gene Expression: The process by which DNA controls the production of proteins via an RNA messenger.

• Central Dogma: Describes the flow of genetic information: DNA → RNA → Protein.

• Genotype: The DNA sequence (e.g., A, T, C, G) of an organism.

• Phenotype: The observable traits, determined by the proteins produced.

Exceptions to the Central Dogma

• Non-coding RNAs: Some genes produce RNA molecules (e.g., tRNA, rRNA) that are not translated into proteins.

• Reverse Transcription: In some viruses, information flows from RNA back to DNA via the enzyme reverse transcriptase.

Transcription: DNA to RNA

Definition and Process

Transcription is the synthesis of RNA from a DNA template. It is the first step in gene expression and occurs in the nucleus of eukaryotic cells and the cytoplasm of prokaryotes.

• RNA Polymerase: The enzyme that catalyzes RNA synthesis by unwinding DNA and adding RNA nucleotides.

• Template Strand: The DNA strand used as a template for RNA synthesis.

• Coding Strand: The non-template DNA strand; the new mRNA is a copy of this strand, except uracil (U) replaces thymine (T).

• Promoter: A special DNA sequence recognized by RNA polymerase to initiate transcription.

• Prokaryotic RNA Polymerase: Composed of a core enzyme (synthesizes RNA) and a sigma subunit (guides the enzyme to the promoter).

• Eukaryotic RNA Polymerases: Three types (I, II, III); RNA polymerase II transcribes mRNA.

• TATA Box: A common promoter sequence in eukaryotes.

• Basal Transcription Factors: Proteins in eukaryotes that help RNA polymerase bind to the promoter.

Differences Between Prokaryotic and Eukaryotic Transcription

• Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm; no nucleus.

• Eukaryotes: Transcription occurs in the nucleus; mRNA is processed (splicing, capping, poly-A tail) before translation.

RNA Processing in Eukaryotes

Splicing and mRNA Maturation

In eukaryotes, the initial RNA transcript (pre-mRNA) contains both coding (exons) and non-coding (introns) regions. Introns are removed by splicing to produce mature mRNA.

• Exons: Coding regions retained in mature mRNA.

• Introns: Non-coding regions removed during splicing.

• Spliceosome: A complex of small nuclear ribonucleoproteins (snRNPs) that removes introns.

• Additional Processing: Addition of a 5' cap and a 3' poly-A tail to stabilize mRNA.

Translation: RNA to Protein

Definition and Mechanism

Translation is the process of synthesizing a protein from an mRNA template. It occurs in the ribosome and involves decoding the mRNA sequence into a polypeptide chain.

• Ribosomes: The sites of translation, composed of rRNA and proteins. Eukaryotic ribosomes have 60S and 40S subunits; prokaryotic ribosomes have 50S and 30S subunits.

• mRNA: Carries the genetic code from DNA to the ribosome; read in the 5' to 3' direction.

• Codon: A sequence of three nucleotides in mRNA that codes for one amino acid.

• tRNA: Transfer RNA molecules carry specific amino acids and have anticodons that pair with mRNA codons.

• Aminoacyl tRNA Synthetase: Enzyme that attaches amino acids to their corresponding tRNA.

• Peptidyl Transferase: Enzyme activity within the ribosome that forms peptide bonds between amino acids.

• Wobble: Non-standard base pairing at the third position of the codon, allowing for redundancy in the genetic code.

Ribosome Sites

• A Site: Accepts new tRNA carrying an amino acid.

• P Site: Holds the tRNA with the growing polypeptide chain.

• E Site: Where tRNA exits the ribosome.

Phases of Translation

1. Initiation: Ribosome binds to mRNA; initiator tRNA (carrying methionine) binds to the start codon.

2. Elongation: Ribosome moves along mRNA, adding amino acids to the growing chain; each peptide bond formation requires energy ($4$ phosphate bonds per peptide bond, mainly from GTP).

3. Termination: Ribosome encounters a stop codon; release factor binds, releasing the new protein and disassembling the ribosomal complex.

Mutations and Their Effects

Types of Mutations

• Substitution: One base is replaced by another (e.g., sickle cell anemia).

• Insertion/Deletion: Addition or removal of bases, causing frameshift mutations.

• Silent Mutation: No effect on the amino acid sequence due to genetic code redundancy.

• Missense Mutation: Changes one amino acid to another.

• Nonsense Mutation: Converts an amino acid codon into a stop codon, often resulting in a nonfunctional protein.

Chromatin Remodeling and Regulation

Differences in Gene Regulation

• Bacteria: Little DNA packaging; sigma factor and promoter regulate transcription; minimal RNA processing.

• Eukaryotes: Chromatin must be opened for transcription; mediator complex and basal transcription factors regulate transcription; extensive RNA processing (splicing, capping, poly-A tail); mRNA stability regulated by RNAi and other mechanisms.

One Gene-One Polypeptide Hypothesis

Historical Experiment: Beadle and Tatum

Beadle and Tatum's experiments with bread mold demonstrated that each gene encodes a specific enzyme. Later research showed that many proteins are composed of multiple polypeptides, each encoded by a separate gene.

• Original Hypothesis: One gene-one enzyme.

• Revised Hypothesis: One gene-one polypeptide.

Table: Beadle and Tatum's Experiment Results

The following table summarizes the results of Beadle and Tatum's experiments, showing the relationship between gene mutations and the ability to synthesize arginine.

Class	Gene Mutated	Growth on Minimal Medium	Growth with Ornithine	Growth with Citrulline	Growth with Arginine
Wild Type	None	Yes	Yes	Yes	Yes
Class I Mutant	Gene 1	No	Yes	Yes	Yes
Class II Mutant	Gene 2	No	No	Yes	Yes
Class III Mutant	Gene 3	No	No	No	Yes

Additional info: This table demonstrates that each gene is responsible for a specific step in the arginine biosynthesis pathway.

Comparison of DNA and RNA

Feature	DNA	RNA
Sugar	Deoxyribose	Ribose
Pyrimidine Bases	Thymine	Uracil
Strandedness	Double stranded	Single stranded

Summary of Key Terms

• Gene Expression: DNA → RNA → Protein

• Transcription: DNA → RNA

• Translation: RNA → Protein

• Mutation: Change in DNA sequence

• Splicing: Removal of introns from pre-mRNA

• Codon: Three-nucleotide sequence in mRNA

• Anticodon: Three-nucleotide sequence in tRNA

Important Equations

• Energy Requirement for Translation: $\text{Energy per peptide bond} = 4 \times \text{phosphate bonds}$

Examples of Protein Types

• Hemoglobin

• Collagen

• Ovalbumin

• Antibodies

• Actin

Additional info: These proteins serve various structural, transport, and immune functions in the body.