Structural Biochemistry/Single-molecule study of RNAP

Transcription Elongation Complex (TEC)
To start transcription, RNA Polymerase (RNAP) must recognize and bind to a promoter sequence. Some factors include assisting the polymerase to an open promoter complex in which the DNA exposes the bases, forming a transcription bubble. Then, RNAP typically undergoes an abortive initiation in which the process synthesizes short strands of RNA transcripts. RNAP returns to the initial promoter site and escapes the region by forming a stable, transcription elongation complex (TEC) which is able to transcribe the whole gene.

Atomic Force Microscopy (AFM)
Atomic force microscopy is a technique used to image the ultrastructural alteration in the TEC such as the change in bend angles of the template DNA induced by RNAP. The TEC is placed on a flat surface then scanned with a AFM cantilever which is a beam anchored at one end. Then, deflections are detected by a laser that reflects the surface. This allows the reconstruction of two-dimensional image of transcriptional complex.

Single Molecule Fluorescence
Another technique used to monitor transcription is to fluorescently tag the RNAP itself. This method allows the monitor of promoter search or elongation with minimal perturbation. Specifically, the structural change in TEC can be examined by using the method called Fluorescence resonance energy transfer (FRET). FRET can follow the distance between two nucleotides by measuring the intensity change in fluorescence.

Beads
By attaching beads to single RNAP molecules, one can record the position of these beads to determine the change in location or rotational state of the enzyme. Specifically, the beads can be sensitively measured by measuring the light scattered from the bead or the rotational states. One can also apply force on the beads with an OT. OT is a tightly focused beam of infrared laser light that exerts forces on the beads by means of radiation pressure. In addition, force can be applied by means of laminar fluid flow. The end of the DNA template can be attached to a second bead so that fluid flow can exert force on the free bead which place tension on the DNA template.

Promoter Search
Transcription requires a binding of the holoenzyme to DNA promoter sequence that is placed throughout an excess of genomic DNA. This is a problem that is common to all sequence-specific DNA-binding proteins. Two independent mechanisms, sliding and intersegment transfer, have been proposed to enhance binding by increasing its efficiency of the search process. Sliding transfer occurs when RNAP associates with nontarget DNA by diffusing in a random “walk” until it reaches the target site. Meanwhile, transegment transfer involves polymerase searching for the promoter by crossing from on position to another, bound simultaneously to both DNA segments.

Open-Complex Formation
When locating a promoter site, the RNAP undergoes a structural transition from the closed complex to the open complex (OPC). The RNAP bends and unwinds a segment of DNA with the aid of initiation factors such as "sigma", creating the transcription bubble. "sigma" is dubbed as the “housekeeping” factor that directs RNAP to recognize vast number of promoters in bacteria. For instance, AFM reading of E. Coli promoter revealed that the DNA is bent between 55̊ and 88̊ which is a consistent measurement from the bend angles inferred from gel mobility assays.

Abortive Initiation
After forming OPC, RNAP starts the synthesis of RNA oligonucleotide complementary to the DNA template strand. Although RNAP creates highly stable complex during elongation phase, the initially transcribing complex (ITC) is highly unstable causing spontaneous release of short RNA chains and restarting synthesis which is known as “abortive initiation.”

On-Pathway Elongation
During transcription, RNAP translocates along the template DNA synthesizing an mRNA that has thousands of nucleotides in length. When the mRNA reaches 9-11nt in length, RNAP leaves the promoter region and enters the elongation phase. In this step, the TEC complex is very stable and remains tightly bound to both the DNA template and the nascent RNA during nucleotide addition. The major stabilizing factor of the complex is thought to be the base pairing within the RNA:DNA hybrid. The “sliding clamp” model states that the extensive protein-nucleic acid contacts within the polymerase greatly contributes to RNA retention, increasing the overall stability. The “clamp” that consists of narrow protein channels surround the hybrid to prevent any shearing motion between the RNA and the DNA.

Off-Pathway Events
The process of on-pathway elongation is frequently interfered by entry into off-pathway states that plays an important role in regulating RNA synthesis. One example of RNA regulation is transcriptional pausing during elongation. The puases can reduce rate of mRNA production, recruit factors for the TEC that modify the subsequent transcription, function as a precursor to termination, or lead to messenger splicing. The long “stabilized” pauses are known to play a regulatory role in formation of RNA hairpins in the transcript which is thought to inactivate RNAP. Series of studies have displayed that pauses lasting 20 seconds or more indicates a rate of base misincorporation during RNA synthesis, suggesting in need for proofreading.

Termination
Termination is a tricky step because of the stability of the TEC complex and RNAP must dissociate accurately releasing the mRNA and the DNA template. In prokaryotes, the termination occurs at specific sequence that code for a stable hairpin in the nascent RNA. In general, termination might be caused through allosteric interactions between RNA hairpin and RNAP that trigger the TEC to release the substrates to stop the reaction. Some studies concluded that termination occurs due to an intermediate elongation-incompetent state whereas some studies support that termination occurs rather quickly with no intermediates.

Reference
Herbert, Kristina M., William J. Greenleaf, and Steven M. Block. "Single-Molecule Studies of RNA Polymerase: Motoring Along." Annual Review of Biochemistry 77.149-76 (2008): 149-172. Print.