Study Notes on Post-Transcriptional Regulation

Post-Transcriptional Regulation

Overview

  • Focuses on how the regulation of gene expression occurs after transcription, impacting the mRNA produced.

Attenuation

  • Definition: Attenuation is a regulatory mechanism that decreases the level of mRNA production when tryptophan is abundant.

  • Mechanism of Action:

    • Attenuation occurs after transcription initiation.

    • Mutations that reduced or eliminated attenuation have been mapped to a specific region between the operator and the trpE gene, which is now referred to as the leader sequence.

    • The leader sequence is 160 base pairs long and located at the 5’ end of the mRNA.

Mechanism of Attenuation

  • Bacterial Characteristics: Bacteria do not have a membrane-bound nucleus, allowing ribosomes to initiate translation while transcription is underway.

  • Without a repressor:

    • Translation starts with the ribosome binding to the mRNA.

    • The process halts after the first 140 base pairs if tryptophan is present.

  • The leader sequence encodes a 14-amino acid peptide, which includes two codons for tryptophan, the least abundant amino acid.

  • If the leader sequence is fully translated:

    • It can create a stem-loop structure in the mRNA, termed a riboswitch.

    • The riboswitch can have two possible conformations depending on the presence of tryptophan.

Diagram Representation

  • Depicts the process of attenuation:

    1. **High Tryptophan Levels:

    • Ribosome fully translates the leader region.

    • The mRNA forms a stem-loop structure leading to transcription termination.**

    1. **Low Tryptophan Levels:

    • Ribosome stalls at tryptophan codons.

    • Transcription continues uninterrupted.**

  • This process is demonstrated visually in resources like animations available in educational literature.

Example: Bacillus subtilis TRAP Attenuator Mechanism

  • TRAP (Tryptophan Activator Protein):

    • Acts as a repressor (Mtr-B) that binds to the leader sequence of the mRNA and promotes the formation of a terminator structure only in the presence of tryptophan.

    • Mechanism:

    • If tryptophan is present, it binds to the Mtr-B protein TRAP, thereby activating it.

    • TRAP induces the formation of a terminator stem-loop structure, leading to transcription termination.

  • TRAP characteristics:

    • Multimeric protein composed of 11 subunits, each capable of binding one molecule of tryptophan.

    • Activation causes TRAP to become circularly oriented.

    • The mRNA is wrapped by the protein complex, resulting in transcription termination.

    • TRAP binds to four sites in B. subtilis resulting in regulatory control of at least four operons, including the folate and mtr operons, forming a Regulon.

Riboswitches

  • Definition: Segments of RNA located in the 5’ untranslated regions (UTR) of mRNAs, which form a three-dimensional structure that can bind small ligands.

  • Small Ligands Examples:

    • Vitamins: Riboflavin, Thiamine, Cobalamin.

    • Amino Acids: Methionine, Lysine.

    • Purine Nucleotides: Adenine, Guanine.

  • Mechanism:

    • Binding of ligands induces changes to the secondary structure of the mRNA riboswitch, exerting regulatory functions.

    • Ligand binding stabilizes one conformation, while the absence of a ligand favors an alternative conformation.

Functions of Riboswitches

  • Riboswitches can control:

    • Transcription.

    • Translation.

    • mRNA stability.

  • Their diversity and specificity point towards their possible status as one of the oldest genetic control mechanisms.

  • While most riboswitch studies focus on bacteria, recent research shows riboswitches are present in eukaryotes as well.

  • Riboswitches demonstrate the ability to sense various molecules, including carbohydrates, amino acids, metals, and coenzymes, indicating a wide regulatory scope.

  • Classes of Riboswitches: There are approximately 38 known classes of riboswitches.

  • Components:

    • The aptamer domain of riboswitches is highly conserved across diverse organisms.

    • The selectivity and specificity of riboswitch molecular recognition are comparable to that of protein receptors.

    • Their functionality supports the RNA world hypothesis, indicating an ancient RNA-based world existed before DNA-based organisms.

Ribozymes

  • Definition: RNA molecules with enzymatic properties that can act as enzymes, notably inducing cleavage to prevent translation.

  • Historical Context:

    • The belief that enzymes were exclusively protein-based was challenged following the discoveries by Sidney Altman and Thomas R. Cech, who were awarded the Nobel Prize in Chemistry in 1989 for their findings on the catalytic properties of RNA.

  • Discovery History:

    • While purifying the enzyme for an intron splicing reaction, Cech discovered that RNA could splice itself without external protein factors.

    • Other studies unveiled self-cleaving RNA or catalytic RNA.

  • Structural Features:

    • Many ribozymes possess hairpin or hammerhead shaped active sites.

    • Their unique secondary structures enable specific RNA cleavage at given sequences.

  • Ribozymes are now recognized for their ubiquity across different biological systems.