In gene expression, promoter-proximal pausing is a widespread regulatory checkpoint. The enzyme RNA Polymerase II (Pol II), responsible for transcribing DNA into RNA, temporarily stops just after it begins its work. This pause occurs on many genes in multicellular organisms, serving as a control point to fine-tune the flow of genetic information. This allows cells to mount swift and coordinated responses to various signals.
The Pausing Mechanism
The process begins when RNA Polymerase II assembles at a gene’s promoter. After Pol II begins to create a new RNA molecule, it travels a short distance, typically 20 to 60 nucleotides, before it halts. This stop is not accidental but is actively caused by specific proteins that act as a brake. Two main factors are responsible for initiating and maintaining this paused state.
The first is the DRB Sensitivity-Inducing Factor (DSIF), which binds to Pol II shortly after transcription begins. Following DSIF’s association, the Negative Elongation Factor (NELF) complex is recruited. NELF works with DSIF to lock the polymerase in place, preventing it from moving further along the DNA. The interaction between DSIF, NELF, and Pol II forms a stable, paused complex that holds the gene in a state of suspended animation.
This molecular brake ensures that the polymerase remains at the ready. The structure of this paused complex has been visualized using advanced techniques like cryo-electron microscopy, revealing the precise interactions that keep the polymerase stalled. The presence of the paused Pol II also helps to keep the chromatin—the packaged structure of DNA—in an open and accessible state around the promoter, making it easier for other regulatory proteins to access the DNA.
Release and Elongation
For a paused gene to become fully active, the molecular brakes must be released, allowing RNA Polymerase II to resume its journey. This release is triggered by another protein, the Positive Transcription Elongation Factor b (P-TEFb). P-TEFb acts as the primary catalyst for overcoming the pause, functioning as a kinase—an enzyme that adds phosphate groups to other proteins.
The catalytic component of P-TEFb, a subunit called Cdk9, is responsible for phosphorylating several targets. One of its first actions is to add phosphate groups to the NELF complex. This modification causes NELF to lose its grip on the polymerase and detach, removing one of the main components of the brake.
Simultaneously, P-TEFb phosphorylates the DSIF protein. This changes DSIF’s function from a repressive factor into a processive factor that assists in elongation. Instead of holding Pol II back, the phosphorylated DSIF now helps it move forward more efficiently.
A final target of P-TEFb is the RNA Polymerase II enzyme itself. P-TEFb phosphorylates a specific region of Pol II known as the C-terminal domain (CTD). This phosphorylation serves as a signal for the polymerase to transition from its paused state into the productive elongation phase, allowing for the synthesis of a full-length RNA molecule.
Regulatory Functions of Pausing
Promoter-proximal pausing provides cells with a mechanism for controlling gene expression with precision and speed. By holding RNA Polymerase II in a paused state, genes are kept in a “poised” condition, ready for immediate activation. This allows for a rapid and synchronized transcriptional response, which is important for genes involved in developmental processes and responses to environmental signals.
A clear illustration of this function is seen with heat shock genes. Under normal conditions, these genes have Pol II paused near their promoters. When a cell is exposed to heat stress, P-TEFb is rapidly recruited to these genes, releasing the paused polymerase and leading to a burst of transcription. This allows the cell to quickly produce the protective proteins needed to survive.
Pausing also functions as a checkpoint, providing a delay between the initiation of transcription and the commitment of cellular resources. During this pause, the cell has an opportunity to integrate multiple regulatory signals, ensuring that the decision to express a gene is made only after all necessary conditions are met.
This checkpoint also allows for quality control. The pause provides time for the proper capping of the newly synthesized RNA molecule, a modification important for its stability and subsequent translation. The polymerase will only be released to continue transcription once this capping process is successfully completed, linking RNA processing to transcription regulation.
Implications in Health and Disease
The precise control of promoter-proximal pausing is fundamental for normal cellular function, and its disruption can contribute to various diseases. The misregulation of this process can lead to the inappropriate expression or silencing of genes, with consequences evident in cancer and viral infections where the pausing machinery is often dysregulated.
In many cancers, the regulation of pause release is altered, leading to the overexpression of oncogenes—genes that can cause cells to become cancerous. The transcription factor c-MYC, which is often hyperactive in cancer cells, can recruit P-TEFb to genes, forcing the release of paused Pol II and driving uncontrolled cell proliferation. Conversely, the failure to release paused Pol II at tumor suppressor genes can lead to their silencing.
The pausing mechanism is also exploited by certain viruses. A prominent example is the Human Immunodeficiency Virus (HIV), which can enter a latent state within host cells. HIV achieves this latency by integrating its genetic material into the host genome and allowing Pol II to pause on its viral promoter. To reactivate, the virus produces a protein called Tat, which hijacks the host cell’s P-TEFb to overcome the pause and drive the production of new viral particles.