Gene Expression at the Molecular Level: Production of mRNAs and Proteins

Chapter 12: 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

  • Levels of Gene Function

    • Molecular function of the protein product

    • Organism’s trait conferred by the gene

    • The connection:

    • Molecular function affects the structure and function of cells, determining the trait.

  • Historical Context

    • The realization that most genes store information to make proteins and the molecular steps of gene expression.

    • Focus on protein-coding genes that encode polypeptides; non-coding RNAs (ncRNAs) will be addressed in the next chapter.

Mutations

  • Historical inquiry before the identification of DNA as genetic material:

    • “How do genes produce the traits of living organisms?”

  • Definitions:

    • Mutations: Changes in genetic material that can be passed from cell to cell and/or from parent to offspring; these alter gene function.

Inborn Errors of Metabolism

  • 1908: Archbold Garrod proposed the link between genes and enzyme production.

  • Studied patients with "inborn errors of metabolism" such as:

    • Alkaptonuria: Accumulation of homogentisic acid; recessive inheritance pattern.

    • Hypothesis: “Disease is due to a missing enzyme.”

Phenylalanine Metabolism

  • Mutations can cause metabolic disorders such as:

    • Phenylketonuria (caused by lack of phenylalanine hydroxylase)

    • Tyrosinosis (caused by lack of hydroxyphenylpyruvate oxidase)

    • Alkaptonuria (caused by lack of homogentisic acid oxidase)

Beadle and Tatum

  • Early 1940s: Notable research with Neurospora crassa (bread mold).

  • Studied minimal growth requirements:

    • Carbon source (sugar), inorganic salts, biotin

    • Neurospora synthesizes other needed compounds from these.

  • Phenomenon:

    • Wildtype strains could grow on minimal medium, but mutant strains required supplementation with specific nutrients (vitamins/amino acids).

    • Formulated the hypothesis: “One gene, one enzyme.”

Experimental Findings

  • Multiple mutants requiring arginine could be grouped based on growth requirements, reinforcing the “one gene, one enzyme” hypothesis.

Modern Understanding

  • Modifications of the “one gene, one enzyme” hypothesis:

    1. Many proteins do not function as enzymes.

    2. Some proteins consist of two or more polypeptides.

    3. Alternative splicing can produce more than one polypeptide from a single gene.

    4. Some genes produce non-coding RNAs that do not translate to polypeptides.

The Central Dogma

  • Processes

    • Transcription: Produces mRNA from DNA, specifying the amino acid sequence of a polypeptide.

    • Translation: Synthesis of the polypeptide on a ribosome using the mRNA template.

  • General Concept:
    ext{DNA}
    ightarrow ext{RNA}
    ightarrow ext{Protein}

  • Differences between Eukaryotes and Prokaryotes:

    • In eukaryotes:

    • Transcription takes place in the nucleus; translation in the cytosol.

    • Includes an additional step: RNA modification.

    • In bacteria:

    • Both transcription and translation occur in the cytoplasm.

Molecular Gene Expression Comparison
Function of Genes

  • Genes are the genetic material, acting as a "blueprint" for organism characteristics.

  • Protein-coding genes store information for producing polypeptides.

  • Polypeptides (as proteins) influence cell structure and function, hence affecting traits of an organism.

Transcription

  • Definition: An organized unit of base sequences enabling a segment of DNA to be transcribed into RNA, forming a functional product such as mRNA, which specifies the amino acid sequence of a protein.

  • Other gene products include:

    • tRNA: Translates mRNA into amino acids.

    • rRNA: Structural component of ribosomes.

Organization of Protein-Coding Genes

  • Promoter: Controls when and where transcription begins.

  • Terminator: Marks the end of transcription.

  • Regulatory Sequences: Sites for binding of regulatory proteins influencing transcription rates.

Gene Structure

Transcription Stages

  1. Initiation

    • Recognition: Sigma factors in bacteria help RNA polymerase find the promoter region; formation of open complex commenced.

  2. Elongation

    • RNA polymerase synthesizes RNA using the template strand, generating mRNA in the 5' to 3' direction.

    • Complementary pairing: The template strand is read 3' to 5'.

  3. Termination

    • RNA polymerase reaches the terminator, leading to dissociation of both the transcript and RNA polymerase from the DNA.

Transcription Stages

Eukaryotic vs Prokaryotic Transcription

  • Eukaryotic cells have more complex transcription mechanisms:

    • Employ three forms of RNA polymerase (RNA polymerase I, II, and III).

    • RNA polymerase II transcribes mRNA and requires six general transcription factors to form a preinitiation complex.

RNA Modification in Eukaryotes

  • Processing of Pre-mRNA

    • Bacterial mRNAs are immediately translated; eukaryotic pre-mRNAs require processing.

    • Introns: Non-coding sequences removed during splicing.

    • Exons: Coding sequences retained.

  • Splicing: Removal of introns occurs; exons are connected, yielding mature mRNA.

Capping and Tailing

  • Capping: Addition of 7-methylguanosine at the 5' end; necessary for nuclear export and ribosome binding.

  • Poly-A Tail: 100-200 adenine nucleotides added to the 3' end, assisting in mRNA stability and lifespan in the cytosol.

RNA Modification Process

RNA Splicing

  1. Definition: Introns are sequences not translated, whereas exons are part of mature mRNA.

  2. Spliceosome: A complex of snRNPs (small nuclear ribonucleoproteins) that facilitates intron removal and exon connection.

  3. Catalysis: The RNA in one of the snRNPs acts as a ribozyme (a ribonucleic acid with enzymatic functions).

Alternative Splicing

  • The process allows a single gene to encode multiple polypeptides, enhancing protein diversity.

Translation and the Genetic Code

  • Genetic Code: Specifies relationships between mRNA bases and amino acids, read in triplets (codons).

  • Codons correspond to amino acids, including Start and Stop codons.

  • Degenerate Code: More than one codon can specify the same amino acid.

Genetic Code Table

Bacterial mRNA

  • Features a 5′ ribosomal-binding site and the start codon (AUG); coding sequence spans from START to STOP codon.

Codons and Anticodons

  • Codon: A triplet of nucleotide bases in mRNA.

  • Anticodon: A sequence in tRNA that pairs with the corresponding mRNA codon.

Polypeptide Synthesis Direction

  • Polypeptide synthesis follows the 5′ to 3′ orientation of mRNA, starting with the amino end (N-terminus) and finishing at the carboxyl end (C-terminus).

Anticodon-Codon Recognition

  • Example: If a tRNA anticodon sequence is 3′–CAG–5′, it pairs with an mRNA codon sequence of 5′–GUC–3′, specifying the amino acid valine.

Synthetic RNA Experiments

  • Biochemists established the genetic code using an in vitro translation system;

    • Nirenberg and Ochoa synthesized mRNA to identify incorporated amino acids in polypeptides.

Translation Machinery

  • Components required:

    • mRNA: Information for the amino acid sequence.

    • tRNA: Binds to mRNA codons and carries amino acids.

    • Ribosomes: Catalyze the formation of polypeptides.

    • Translation Factors: Assist in initiation, elongation, and termination stages.

Translation Machinery

tRNA Structure and Function

  • tRNA has a cloverleaf structure utilizing an anticodon and an acceptor stem for amino acid binding.

  • Each amino acid has a corresponding aminoacyl-tRNA synthetase for attachment to the tRNA.

Ribosomes

  • Sites for translation; distinct structures in prokaryotes (70S) and eukaryotes (80S).

  • Composed of large and small subunits; sites for tRNA binding (A, P, E) exist on the ribosome.

Evolutionary Relationships

  • Small subunit rRNAs give insight into evolutionary history and relationships among species by analyzing sequences.

Stages of Translation

  1. Initiation: Assembly of mRNA, first tRNA, and ribosomes.

  2. Elongation: Synthesis from start codon to stop codon.

  3. Termination: Components disassemble at the stop codon releasing the polypeptide.

Stages of Translation