MACEE CHAPTER 12 BIO110

Gene Expression at the Molecular Level

Key Concepts

  • Overview of Gene Expression

  • Transcription

  • RNA Modification in Eukaryotes

  • Translation and the Genetic Code

  • The Machinery of Translation

  • The Stages of Translation

Overview of Gene Expression

  • Gene function can be analyzed at two interconnected levels:

    • Molecular function of the protein product

    • Trait of the organism conferred by the gene

  • The molecular function influences the structure and function of cells, thereby determining the organism's traits.

How do Genes Determine Traits?

  • Genes are the genetic material, serving as blueprints for the characteristics of organisms.

  • Structural Genes: These genes code for polypeptides, which combine to form proteins.

  • Proteins play crucial roles in cells, determining structure and function.

  • Collectively, the activities of proteins in an organism dictate its traits or characteristics.

Inborn Errors of Metabolism

  • In 1908, Archbold Garrod theorized the link between genes and enzyme production.

    • Observed patients with metabolic defects, termed "inborn errors of metabolism."

    • Example: Alkaptonuria - A condition where homogentisic acid accumulates due to a defective enzyme.

  • Hypothesis: "Disease is due to a missing enzyme."

  • Phenylketonuria: Mutation causes the inability to convert phenylalanine (found in diet sodas) to the next chemical in the metabolic pathway.

  • Tyrosinosis: Another condition caused by enzyme mutations affecting metabolic pathways.

Beadle and Tatum's Experiment (1940s)

  • Beadle and Tatum studied Neurospora crassa, a common bread mold.

    • Wildtype Neurospora: Can grow on minimal medium (sugar and inorganic salts).

    • Growth Requirements: Minimal medium needs carbon sources, inorganic salts, and biotin.

Simplified Pathway for Arginine Synthesis

  • Wildtype Neurospora synthesizes arginine in 3 steps, requiring three different enzymes.

Creation of Mutant Strains

  • Mutant Strains: Strains 1, 2, and 3 unable to grow without specific nutrient supplementation (vitamins or amino acids).

    • Each mutation corresponds to a specific nutrient requirement.

  • Mutant strains can grow if supplemented with the product of the enzyme they lack or downstream molecules in the pathway:

    • Group 1: Deficient in enzyme 1, can utilize ornithine, citrulline, or arginine.

    • Group 2: Deficient in enzyme 2, can utilize citrulline or arginine.

    • Group 3: Deficient in enzyme 3, can only utilize arginine.

Modern Understanding of Genes and Enzymes

  • The original "one gene, one enzyme" hypothesis evolved:

    • Enzymes are just a subset of cellular proteins;

    • Genes can encode multiple types of proteins and proteins can consist of multiple polypeptides.

    • Example: Hemoglobin consists of 4 polypeptides.

The Central Dogma of Gene Expression

  • Transcription: Generates an RNA copy of a gene (transcript).

  • Types of RNA:

    • Messenger RNA (mRNA): Specifies the amino acid sequence of polypeptides.

    • Transfer RNA (tRNA) and Ribosomal RNA (rRNA): Also produced via transcription.

  • The overall expression of a gene occurs via:

    • Gene Expression = Transcription + Translation.

RNA Processing in Eukaryotes

  • Eukaryotic cells possess a distinct processing phase where pre-mRNA is processed into mature mRNA.

  • Not all genes lead to polypeptide production; some encode final functional RNA molecules like tRNA.

Molecular Gene Expression in Prokaryotes vs. Eukaryotes

  • Bacterial Gene Expression:

    • Transcription and translation occur in the cytoplasm as there is no nucleus.

  • Eukaryotic Gene Expression:

    • Transcription and modifications occur within the nucleus; mRNA exits through nuclear pores to the cytosol for translation.

Transcription Process

  • Gene Definition: A gene is an organized unit of DNA that is transcribed into mRNA leading to a functional product, usually a polypeptide.

  • Types of RNA Genes:

    • tRNA: Translators of mRNA into amino acids.

    • rRNA: Integral parts of ribosomes for polypeptide synthesis.

Transcription Regions in a Gene

  1. Regulatory region: Where proteins influence transcription rate.

  2. Promoter: Marks transcription initiation.

  3. Transcribed region: Specifies mRNA and subsequently amino acid sequences.

  4. Terminator: Signals end of transcription.

Stages of Transcription

  1. Initiation:

    • Recognition of the promoter region; RNA polymerase binds with sigma factor to start transcription.

    • DNA strands separate to form an open complex.

  2. Elongation:

    • RNA polymerase synthesizes RNA using the DNA template strand.

    • RNA is synthesized in the 5' to 3' direction; uracil replaces thymine.

  3. Termination:

    • RNA polymerase encounters the termination sequence, causing detachment from DNA and release of the RNA transcript.

Detailed Process of Initiation

  • Promoter serves as the recognition site.

  • Sigma factor helps RNA polymerase bind effectively; DNA unwinds to form an open complex.

Detailed Process of Elongation

  • RNA polymerase synthesizes RNA in the opposite direction of the DNA template strand direction, moving from 5' to 3'.

Detailed Process of Termination

  • Upon reaching the terminator sequence, RNA polymerase and the RNA dissociate, ending transcription.

Eukaryotic Transcription Elements

  • Eukaryotic transcription maintains foundational similarities with prokaryotic; however, involves more proteins and has distinct RNA polymerases.

    • RNA Polymerase II: Transcribes mRNA.

    • RNA Polymerase I and III: Transcribe nonstructural genes for rRNA and tRNA.

  • Requires 5 general transcription factors to begin.

RNA Modification in Eukaryotes

  • Eukaryotic mRNAs must undergo processing to become mature mRNA.

    • Introns: Non-coding regions that are transcribed but not translated (removed during splicing).

    • Exons: Coding sequences that appear in mature mRNA.

Key Steps in RNA Processing/Modification

  1. Splicing: Removal of introns to join exons.

  2. Capping: A modified guanosine cap is added to the 5' end for nuclear exit and ribosome binding.

  3. Poly A Tail Addition: 100 to 200 adenine nucleotides added to the 3' end for increased stability in the cytosol (not part of the gene sequence).

Spliceosome Mechanism

  • A spliceosome composed of snRNPs (small nuclear RNA + proteins) precisely removes introns during splicing.

Translation and the Genetic Code

  • The Genetic Code consists of sequenced bases in an mRNA molecule, which are read in groups of three bases (codons).

  • Most codons correspond to specific amino acids and comprise start and stop signals.

  • The genetic code is degenerate, meaning multiple codons can code for the same amino acid.

Bacterial mRNA Structure

  • Contains a 5' ribosomal-binding site and a start codon (typically AUG).

  • Coding sequence contains several codons that encode polypeptides, concluded by stop codons (UAA, UAG, UGA).

Codons and Anticodons

  • A codon in mRNA corresponds to an amino acid and involves a substitution of T with U.

  • Anticodon: The complimentary 3-nucleotide sequence on tRNA that binds to the mRNA codon.

Reading Frame and Mutations

  • The reading frame is determined by the start codon, and any mutations can cause shifts in the reading frame, affecting translation.

  • Example: Addition of a U impacting initial sequences specified.

Sequence Details in Translation Process

  • Involves codons from the mRNA, anticodons from tRNA, and corresponding amino acids for polypeptide formation.

Equipment Used in Translation

  • Translation is energy-intensive, requiring components such as:

    • mRNA

    • tRNA

    • Ribosomes

    • Translation factors

Common Features of tRNA

  • Structure includes:

    • Cloverleaf form

    • Anticodon for mRNA binding

    • Acceptor stem for amino acid binding.

Aminoacyl-tRNA Synthetase

  • Enzyme catalyzing the attachment of amino acids to tRNA; specific for each of the 20 amino acids, resulting in a charged tRNA.

Components of the Translation Machinery

  • mRNA: Contains information for amino acid sequences as per the genetic code.

  • tRNA: Binds to both codons on mRNA and attaches amino acids.

  • Ribosomes: Catalyze formation of covalent bonds between amino acids, facilitating polypeptide synthesis.

  • Translation Factors: Include initiation factors, elongation factors, and release factors to aid translation processes.

Ribosome Assembly

  • Ribosomes consist of rRNA and proteins, with specific sedimentation coefficients denoting their size (70S for prokaryotes and 80S for eukaryotes).

  • Ribosomes feature distinct sites:

    • P site: Peptidyl site

    • A site: Aminoacyl site

    • E site: Exit site.

Three Stages of Translation:

  1. Initiation: mRNA, tRNA, and ribosomal subunits form a complex.

  2. Elongation: Ribosome travels in the 5' to 3' direction, synthesizing the polypeptide.

  3. Termination: Stop codon recognition leads to the disassembly of translation machinery and release of the completed polypeptide.

Detailed Process of Initiation

  • The ribosomal-binding sequence assists in binding mRNA to the small ribosomal subunit, with initiator tRNA locating the start codon.

Detailed Process of Elongation

  • Charged tRNA enters the A site; peptide bonds form at the P site, with each step using GTP energy.

  • Translocation shifts the ribosome for the next codon.

Detailed Process of Termination

  • Stop codons recognized in the A site trigger polypeptide release via activity from release factors; dissociation of ribosomal subunits occurs following synthesis completion.

Antibiotics Targeting Translation

  • Antibiotics that target bacterial translation are effective against bacterial infections due to differences in ribosome structures compared to eukaryotic cells.