Week 9 - L1 - Protein Regulation via Post-Translational Modifications and RNA Polymerase II CTD Dynamics

Overview of Post-Translational Modifications and Disease

  • Scope of Gene Expression:

    • Gene expression encompasses the full biochemical pathway extending from the gene to the fully functional protein.

    • The completion of translation—synthesizing the complete polypeptide chain—does not mark the endpoint of gene expression.

    • Proteins typically require post-translational modifications (PTMs) to achieve full biological functionality.

  • Mechanisms and Types of Post-Translational Modifications:

    • PTMs involve the covalent attachment of chemical groups, complex molecules, or entire proteins to amino acid residues.

    • Small Chemical Groups:

      • Hydroxyl groups (-OH\text{-OH})

      • Methyl groups (-CH3\text{-CH}_3)

      • Acetyl groups (-COCH3\text{-COCH}_3)

      • Phosphate groups (-PO43−\text{-PO}_4^{3-})

    • Macromolecules and Lipids:

      • Lipids (lipidation)

      • Complex sugars (glycosylation)

    • Whole Protein Modifications:

      • Ubiquitin (polyubiquitylation): Serves as a signal for loss of function and marks target proteins for degradation.

  • Functional States and Pathological Implications:

    • PTMs dynamically regulate protein function by mediating transitions between different functional states.

    • Dysregulation of PTM pathways—caused by mutations or aberrant enzymatic activity—alters normal modification patterns and leads to disease states.

Structure and Function of the Eukaryotic RNA Polymerase II Carboxyl Tail Domain

  • Role of RNA Polymerase II:

    • Synthesizes eukaryotic messenger RNA (mRNA) from protein-coding genes.

    • Directly coordinates co-transcriptional processing of the primary RNA transcript.

  • Carboxyl Tail Domain (CTD) Spatial Positioning and Function:

    • Serves as the central control center for orchestrating mRNA processing.

    • Strategically located near the exit site where nascent mRNA emerges from the RNA polymerase II enzyme complex.

    • Organizes the sequential recruitment and release of processing machinery:

      1. 5' Capping enzymes

      2. Spliceosome factors

      3. 3' Polyadenylation factors

  • Structural Properties of the CTD:

    • The CTD is intrinsically disordered (unfolded).

    • Its unfolded structure provides maximal accessibility for regulatory enzymes:

      • Kinases: Enzymes that covalently add phosphate groups.

      • Phosphatases: Enzymes that remove phosphate groups.

  • Consensus Sequence and Functional Residues:

    • Consists of repeating units of a seven-amino-acid consensus sequence: tyrosine, serine, proline, threonine, serine, proline, serine.

    • Each heptapeptide repeat contains three serine residues:

      • Serine 2\text{Serine } 2

      • Serine 5\text{Serine } 5

      • Serine 7\text{Serine } 7

    • Empirical evidence demonstrates that while all three serines can undergo phosphorylation, only Serine 2\text{Serine } 2 and Serine 5\text{Serine } 5 directly control the expression and processing of protein-coding genes.

Dynamic CTD Phosphorylation Cycle During Transcription and RNA Processing

  • Unbound / Re-initiation State:

    • When RNA polymerase II is not bound to DNA, the CTD is entirely unphosphorylated.

    • Only the completely dephosphorylated form of RNA polymerase II can bind to a promoter to initiate transcription for a new gene.

  • Transcription Initiation and 5' Capping:

    • Upon promoter binding and transcription initiation, the CTD becomes heavily phosphorylated on serine residues.

    • A transcription factor recruited to the promoter contains intrinsic kinase activity and specifically phosphorylates Serine 5\text{Serine } 5.

    • 5' Capping enzymes specifically recognize and bind to the CTD when it is phosphorylated on Serine 5\text{Serine } 5.

    • This spatial and temporal mechanism ensures the 5' end of the mRNA is capped as soon as it exits RNA polymerase II.

  • Transcription Elongation and Splicing:

    • As RNA polymerase II transcribes further into the gene body, a distinct kinase associated with the elongating polymerase extensively phosphorylates Serine 2\text{Serine } 2.

    • This dual-phosphorylation state is recognized by splicing factors.

    • The spliceosome complex is recruited directly to the CTD to carry out co-transcriptional RNA splicing.

  • Transcription Termination and 3' Polyadenylation:

    • As RNA polymerase II approaches the end of the gene, phosphatases remove phosphate groups specifically from Serine 5\text{Serine } 5 residues.

    • This creates a new phosphorylation profile featuring phosphorylated Serine 2\text{Serine } 2 and unphosphorylated Serine 5\text{Serine } 5

    • 3' End processing proteins recognize this specific pattern, bind to RNA polymerase II, and travel with it until the polyadenylation (poly-A) signal is transcribed.

  • Transcriptional Completion and Enzyme Recycling:

    • Once gene transcription completes and RNA polymerase II dissociates from DNA, cellular phosphatases remove all remaining phosphate groups from the CTD.

    • The fully dephosphorylated RNA polymerase II returns to its original state, enabling it to bind to a promoter and re-initiate mRNA synthesis.