E. coli RfaH

Paula Cancelas Calvo '22 and Christopher Ponne '22

Contents:

I. Introduction


The Escherichia coli RfaH protein is a universally conserved transcription factor of the NusG/Spt5 family and has only been studied in Escherichia coli. This family of proteins are the only transcription factors that have coevolved with RNAP since the last universal common ancestor. They also have a unique method to recruit and elongate RNA polymerase (RNAP) for the transcription of virulence genes. RfaH, in particular, is found to activate long operons that encode antibiotics, capsules, toxins, and pili through inhibition of Rho-dependent termination (Zuber, 2018).

When RNAP encounters transcriptional pausing or termination, RfaH will work as an antiterminator to assist in elongation. RfaH binds directly to the ops sequence located in the non-template (NT) DNA strand of the transcription bubble. Contact with the ops sequence is thought to trigger domain dissociation, transforming RfaH into an open, active state where the N-terminal domain can bind to RNAP and the C-terminal domain changes structure. Once activated, RfaH remains bound to the transcription elongation complex (TEC) until termination. The CTD recruits the 30S subunit of the ribosome to the template/leader sequence where the ribosome will then translate the RNA after transcription. After TEC dissociation, RfaH regains the autoinhibited state and the cycle repeats (Zuber, 2018). By understanding this protein it may be possible to inhibit RfaH and enable termination of viral genes found in any cell, including human cells.  

RfaHLifeCycle

Figure 1. RfaH recruitment to the ops-paused RNAP via the hairpin loop in the NT strand of the ops sequence. RfaH activates and upon release the CTD changes into a beta barrel. The CTD interacts with the small subunit of the ribosome to begin translation of the RNA afterwards, RfaH is finally released along with RNAP. The process repeats with another ops sequence (Zuber, 2018).

II. General Structure


Proteins of the NusG/Spt5 family usually have N-terminal domains (NTD) of mixed alpha and beta topology connected to beta barrel C-terminal domain (CTD) along with a KOW motif via a flexible linker called the N/C linker. Rfah, however, the CTD consists of two in an alpha helical hairpin that have 2 acidic residues at the tip of the domain ( and ). The NTD binds across the DNA-binding channel, bridging the RNAP beta prime clamp and beta lobe domains pincers and resulting in RNAP becoming pause-resistant. On release, the alpha helical CTD spontaneous forms into a beta barrel (Zuber, 2018)  

III. RfaH and ops interaction


The main difference between NusG and RfaH is that RfaH action depends on the ops site. The ops site is thought to play four roles during RfaH recruitment, first it can slow down RNAP to allow more time for RfaH recruitment. Rfah targets all have a pause-inducing TG dinucleotide at position 11 and 12 (not shown) which allows RNAP delay.Second, it mediates sequence-specific binding of RfaH to the NT DNA strand exposed on the surface of the TEC. Third, induces TEC isomerization into a structurally distinct paused state that may be necessary for productive recruitment of RfaH. Finally, pausing could be required for ribosome recruitment.

T11, as previously mentioned, has an essential role in pausing RfaH activity. Without RfaH, RNAP pauses at C9 and U11 in the ops template strand. With RfaH, pausing at U11 is reduced but increases at G12. However, pausing at U11 is dispensable for RfaH binding when RNAP transcribes slowly (Belogurov, 2007 and Zuber, 2018). It is important to remember that the ops sequence/site is located in the NT hairpin.  

IV. Non-template Hairpin loop interaction with RfaH-NTD


The NT hairpin is required for RfaH recruitment. RfaH-NTD binds to the ops sequence in the NT DNA hairpin loop which forms when the DNA binds to the basic patch of RfaH-NTD, opposite of the RNAP/RfaH-CTD binding site. The loop consists of G4-A7 , with flipped out leaving the nucleobase completely exposed. The other nucleotides of the loop make stacking interactions.

The flipped T6 is inserted into a deep narrow, on Rfah-NTD, which is formed of H20, R23, Q24, and R73 located in helices alpha 1 and alpha 2. is packed in the positive surface next to the cavity T6 is located. RfaH-NTD makes contact with nucleotides in the loop, involving K10, H20, R23, Q24, T68, N70, A71, T72, R73, G74,and V74 . Base specific interactions with RfaH-NTD are made G4, G5, and T6, but only G5 and T6 form a hydrogen bond network and may underline sequence specific recognition. The side chains K10, H20, R23, and R73 directly interact with the ops DNA. There are no aromatic residues located near G5 and T6, therefore contact between these two nucleotides and these 4 amino acids actually mediate specific recognition of ops by RfaH. The hairpin stem is formed by base pairs C3:G8 and G2:C10, with T9 being flipped out (Zuber, 2018).


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Figure 2.

VI. References

Belogurov, G. A., Vassylyeva, M. N., Svetlov, V., Klyuyev, S., Grishin, N.V., Vassylyev, D. G., and Artsimovitch, I. “Structural basis for converting a general transcription factor into an operon-specific virulence regulator.” Molecular cell vol. 26,1 (2007): 117-29.

Kang, J. Y., Mooney, R. A., Nedialkov, Y., Saba, J., Mishanina, T. V., Artsimovitch, I., Landick, R., and Darst, S. A. “Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators.” Cell vol. 173,7 (2018): 1650-1662.e14.

Zuber, P. K., Artsimovitch, I., NandyMazumdar, M., Liu, Z., Nedialkov, Y., Schweimer, K., Rösch, P., and Knauer, S. H. “The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand.” eLife vol. 7 e36349. 9 May. 2018.

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