Gene Expression: The Genetic Code and Transcription

Gene Expression: The Genetic Code and Transcription

• The Genetic Code and the Directional Flow of Genetic Information - Central Dogma of Molecular Biology: The genetic code is fundamental to understanding how genetic information is transferred within a biological system, primarily from DNA to RNA and subsequently to proteins. This process is termed the central dogma of molecular biology.

  • Transcription: This is the first step of gene expression, where a specific segment of DNA is transcribed into messenger RNA (mRNA) by the enzyme RNA polymerase. This process includes stages such as initiation, elongation, and termination.

  • Translation: Following transcription, translation is the process where the mRNA is decoded by a ribosome to synthesize proteins. Each set of three nucleotides (codon) in the mRNA corresponds to a specific amino acid, facilitating the assembly of proteins according to the genetic code.

• Key Implications: Mutations within DNA can significantly influence protein structure and function.

  • 5′ mutations often impact the N-terminal of proteins, which can affect protein stability, localization, or activity.

  • 3′ mutations predominantly influence the C-terminal of proteins, which can lead to alterations in protein degradation rates or interactions with other molecules.

  • The sequence of nucleotides in the mRNA dictates the precise order of amino acids during protein synthesis, determining the functional characteristics of the resulting protein.

  • Notably, the genetic code is considered universal, being largely consistent across different organisms, which highlights evolutionary connections among diverse forms of life.

Transcription and Translation

• Components in both Bacteria and Eukaryotes:

  • mRNA: This molecule functions as a transcript of the gene coding for a specific protein, carrying the genetic instructions from DNA to the ribosome for protein synthesis.

  • rRNA: Ribosomal RNA is a primary component of ribosomes, playing a crucial role in the catalysis of protein synthesis by facilitating the binding of mRNA and tRNA.

  • tRNA: Transfer RNA is responsible for delivering the appropriate amino acids to the ribosome during translation, matching its anticodon to the corresponding codon on the mRNA strand.

The Genetic Code

• Structure: The genetic code consists of triplet codons, and its triplicate nature is essential to accommodate the vast number of amino acids required for protein synthesis.

  • Given that there are four unique DNA bases (adenine, thymine, cytosine, and guanine) and twenty standard amino acids, this necessitates 64 potential triplet combinations to effectively code for all amino acids. Thus, a simple doublet code, which offers only 16 combinations, would be inadequate for this task.

Supporting the Triplet Code Using Frameshift Mutations

• Frameshift Mutations: These mutations arise from the insertion or deletion of nucleotides, which disrupt the reading frame of the mRNA. This alteration can lead to significant changes in the resulting protein.

  • The triplet code theory gained support through various experiments, including those using Proflavin as a mutagen, which caused frameshift mutations and subsequently helped in confirming the coding structure of mRNA.

The Degenerate and Nonoverlapping Nature of the Genetic Code

• Degenerate Code: The genetic code is degenerate, meaning that most amino acids are coded for by more than one codon. This characteristic provides a level of protection against certain mutations, as substitutions may not necessarily impact the resultant protein.

• Nonoverlapping: Each nucleotide in the mRNA is read in groups of three (codons), and these triplets are interpreted independently from one another in a sequential manner without overlapping, ensuring an accurate translation of the genetic information.

Codon Dictionary Establishment

• Homopolymer Experiments: Early experiments involving the use of homopolymeric RNA sequences (like poly-U) were critical in deciphering the codon assignments to specific amino acids, leading to the establishment of a codon dictionary.

• Synthetic RNA Technology: This technological advancement enabled researchers to synthesize RNA molecules, allowing for the detailed studying of codon assignment and helping to elucidate the entire codon dictionary.

Messenger RNA and Its Synthesis

• mRNA Synthesis Differences: The synthesis of messenger RNA involves copying only one strand of the DNA double helix, generating a complementary RNA strand. Notably, uracil is used in place of thymine during this process.

• Four Stages of Transcription:

  1. Binding: RNA polymerase recognizes and binds to the promoter region of the gene, initiating the transcription process.

  2. Initiation: Following binding, the DNA double helix unwinds locally, leading to the first few RNA nucleotides being synthesized.

  3. Elongation: RNA polymerase catalyzes the addition of RNA nucleotides, synthesizing RNA in the 5′ to 3′ direction and adding appropriate ribonucleotides that complement the DNA template.

  4. Termination: Transcription concludes as RNA polymerase reaches specific termination sequences in the DNA, leading to the dissociation of the newly formed RNA molecule from the DNA template.

Complexity in Eukaryotic Transcription

• Three RNA Polymerases: In eukaryotic cells, there are three distinct RNA polymerases (I, II, and III), each responsible for transcribing different types of genes and RNA.

  • RNA Polymerase II

    • INI → Initiator sequence → start and transcription

    • TATA Driven: Moving upstream, Inr, TATA box (may contain BRE)

    • DPE Driven: Moving downstream, Inr (No BRE or TATA)

    • Contains TF II

      • At a TATA-driven promoter, TFIID → binds to TATA box

        • Recruits TFII B → binds to BRE (if present)

      • TFII H → Helicase: unwind DNA at promoter

      • Kinase: Phophorylates c-terminal tail of RNA II (active)

  • RNA Polymerase III

    • Responsible for making tRNA and 5S RNA and moving downstream

    • tRNA genes: Box A and Box B

    • rRNA genes: Box A and Box C

• Promoters: The nature of promoters varies greatly between prokaryotic and eukaryotic systems, with eukaryotic promoters frequently requiring additional transcription factors in order to facilitate the binding of RNA polymerase and initiate transcription effectively.

  • Core promoter is the smallest set of DNA sequences that initiates transcription.

    • Only Basal (low) level of transcription

• General Transcription Factors

  • Bind at promoter

• Specific Transcription Factors

  • Bind control elements

  • Generally upstream of promoter

RNA Processing in Eukaryotes

• Chemical Modifications

  • Removal of neurotransmitters on the ends and in the middle (splicing)

  • Addition of neurotransmitters on the ends

  • Nitrogenous Bases

• Post-Transcriptional Modifications: After transcription, pre-mRNA undergoes several crucial modifications, including capping, polyadenylation, and splicing, to produce a mature mRNA molecule.

• 5′ Cap: This modified guanine nucleotide is added to the 5′ end of the growing mRNA transcript; it plays a vital role in mRNA stability and is important for the initiation of translation by facilitating ribosome binding.

• Poly(A) Tail: A series of adenine nucleotides are added to the 3′ end of mRNA, enhancing its stability and aiding in its recognition during translation.

• Spliceosomes: These complex molecular machines are responsible for RNA splicing, involving small nuclear ribonucleoproteins (snRNPs) to remove introns and join exons together, ultimately producing the functional mRNA.

Alternative Splicing

• Functionality: This molecular mechanism enables a single gene to produce multiple protein variants by employing different splicing patterns, thus contributing to the protein diversity crucial for biological complexity and adaptability in eukaryotic organisms.

Termination of Transcription

• Prokaryotic and Eukaryotic Termination Signals: Termination of transcription varies depending on the type of RNA polymerase. In prokaryotes, termination can occur through intrinsic signals or via Rho-dependent mechanisms, while in eukaryotes, specific sequences lead to the cleavage and subsequent release of the mRNA strand from the polymerase.

  • RNA Pol III

    • Short run of As on template strand

    • UUUUU on RNA strand

  • RNA Pol II

    • Polyadenylation signal sequence is binding site for multiple enzymes

      • Nuclease: cleave RNA with 35 NTs after signal sequence

      • PolyA polymerase: adds the poly A tail (post-transcriptionally)

    • mRNA = AAUAAA for RNA strand

• mRNA Lifespan: Eukaryotic mRNAs generally exhibit a longer half-life compared to bacterial mRNA, impacting how long genetic instructions can be expressed and influencing overall gene expression and regulation.

Amplification of Genetic Information

• Reusability of mRNA: The ability to reuse mRNA allows cells to synthesize multiple proteins from a single DNA template, significantly enhancing gene expression capabilities, as exemplified by the high levels of fibroin gene expression seen in silkworms, which is critical for silk production.