Kinetics of transcription initiation directed by multiplecis-regulatory elements on theglnAp2promoter

Abstract

Transcription initiation is orchestrated by dynamic molecular interactions, with kinetic steps difficult to detect. Utilizing a hybrid method, we aim to unravel essential kinetic steps of transcriptional regulation on the glnAp2 promoter, whose regulatory region includes two enhancers (sites I and II) and three low-affinity sequences (sites III-V), to which the transcriptional activator NtrC binds. By structure reconstruction, we analyze all possible organization architectures of the transcription apparatus (TA). The main regulatory mode involves two NtrC hexamers: one at enhancer II transiently associates with site V such that the other at enhancer I can rapidly approach and catalyze the σ54-RNA polymerase holoenzyme. We build a kinetic model characterizing essential steps of the TA operation; with the known kinetics of the holoenzyme interacting with DNA, this model enables the kinetics beyond technical detection to be determined by fitting the input-output function of the wild-type promoter. The model further quantitatively reproduces transcriptional activities of various mutated promoters. These results reveal different roles played by two enhancers and interpret why the low-affinity elements conditionally enhance or repress transcription. This work presents an integrated dynamic picture of regulated transcription initiation and suggests an evolutionarily conserved characteristic guaranteeing reliable transcriptional response to regulatory signals.

Gene transcription in bursting: a unified mode for realizing accuracy and stochasticity

“…Upstream of the glnAp2 promoter, there are two enhancer sequences and three low-affinity sequences for the binding of transcriptional activator nitrogen regulatory protein C (NtrC). In contrast with the traditional view that transcription initiation is stimulated by an NtrC hexamer simultaneously bound to the two enhancers, Wang et al (2016) revealed that the initiation is stimulated by an NtrC hexamer at either enhancer ( Fig. 3).…”

“…Investigating transcriptional dynamics necessitates both live imaging methods with high resolution (Skupsky et al ., ; Suter et al ., ; Evans et al ., ; Friedman, Mumm, & Gelles, ; Gebhardt et al ., ; Hocine et al ., ; Kouno et al ., ; Lickwar, Mueller, & Lieb, ; Yunger et al ., ; Sidaway‐Lee et al ., ; Annibale & Gratton, ; Camunas‐Soler et al ., ; Gocheva et al ., ; Roberts et al ., ; Rybakova et al ., ; Corrigan et al ., ; Tantale et al ., ) and quantitative computer simulations with appropriate theories and models (Skupsky et al ., ; Suter et al ., ; Wang et al ., ; Maina et al ., ; Choubey, Kondev, & Sanchez, ; Stefan et al ., ; Rybakova et al ., , b ; Corrigan et al ., ; Tantale et al ., ). Specifically, integrating diverse sets of data makes it possible to present a coherent dynamic picture of gene transcription in bacteria (Wang et al ., ).…”

“…In contrast with the traditional view that transcription initiation is stimulated by an NtrC hexamer simultaneously bound to the two enhancers, Wang et al . () revealed that the initiation is stimulated by an NtrC hexamer at either enhancer (Fig. ).…”

“…This is challenging, especially when unstable protein complexes and unknown transient interactions are involved (Friedman & Gelles, 2012). A promising approach is to adopt a hybrid method that combines the data on molecular structure, molecular interaction kinetics, and transcriptional input-output function, as exemplified by a recent study which unraveled the transcriptional dynamics of the glnAp2 promoter (Wang, Liu, & Wang, 2016).…”

Overcoming the Cost of Positive Autoregulation by Accelerating the Response with a Coupled Negative Feedback

“…Similar patterns of strong activation and weak repression sites have also been observed in other autoregulated TCS promoters, such as phoBR in Vibrio cholerae ( Diniz et al, 2011 ) and glnALG in E. coli ( Atkinson et al, 2002 ). Although the mechanism of transcription repression appears to be different as suggested by different positions of repression sites ( Diniz et al, 2011 ; Wang et al, 2016 ), weak repression sites function to similarly limit the maximal concentration at high TF levels. Dual autoregulatory TFs constitute a non-trivial fraction (~10%) of autoregulated TFs in E. coli ( Martínez-Antonio et al, 2008 ), and this number may even be underestimated due to difficulties in uncovering the weak autoregulatory interactions.…”