Regulation of Transcription: Regulation of Transcription Elongation, Pausing, and Termination

Recently we have extended our studies of transcriptional regulation to encompass regulation at the level of transcription antipausing, transcription  and antitermination.

The transcription antitermination factor Q, which is produced by lambdoid bacteriophage during lytic infection, is one of two classic textbook examples of regulators of gene expression that function at the level of transcription pausing and transcription termination (e.g., Molecular Biology of the Gene). (The other classic textbook example is the structurally and mechanistically unrelated regulator N, which is produced by bacteriophage lambda and functions in an earlier phase of lambdoid bacteriophage infection.)

Q proteins function by binding to RNA polymerase-DNA-RNA transcription elongation complexes (TECs) and rendering TECs unable to recognize and respond to transcription pausing and transcription termination signals.  Q proteins are targeted to specific genes through a multi-step binding process entailing formation of a "Q‑loading complex" comprising a Q protein bound to a Q binding element and a sigma-containing TEC paused at an adjacent sigma-dependent pause element, followed by transformation into a "Q-loaded complex" comprising a Q protein and a translocating, pausing‑deficient, termination-deficient TEC.

Q proteins from different lambdoid bacteriophages comprise three different protein families (the Ql family, the Q21 family, and the Q82 family), with no detectable sequence similarity to each other and no detectable sequence similarity to other characterized proteins.  Q proteins from different protein families are thought to be analogs (with identical functions but unrelated structures and origins), rather than homologs (with identical, interchangeable functions and related structures and origins).

Q proteins have been the subject of extensive biochemical and genetic analysis spanning five decades. However, an understanding of the structural and mechanistic basis of transcription antitermination by Q proteins has remained elusive in the absence of three-dimensional structural information for Q‑dependent antitermination complexes.

We are systematically determining high‑resolution single-particle cryo-EM structures of Qlambda-, Q21-, and Q82-dependent transcription antitermination complexes. 

Results for Q21 reveal that Q21 forms a torus--a "nozzle"--that extends and narrows the RNA-exit channel of RNA polymerase, that the nascent RNA is threaded through the Q nozzle, and that the threading of the nascent RNA through the Q nozzle precludes the formation of pause and terminator RNA hairpins.

Narrowing and extending the RNA-exit channel of RNA polymerase by attaching a nozzle and threading RNA through the nozzle is a remarkably straightforward mechanism for antitermination and almost surely will be a generalizable mechanism.

Attaching a nozzle and threading RNA through the nozzle has the additional remarkable consequence of generating a topological connection--an unbreakable linkage--between the antitermination factor and the RNA emerging from RNA polymerase.  This enables exceptionally stable association and exceptionally processive antitermination activity and has implications for engineering highly efficient, tightly regulated, gene expression for synthetic biology applications.