Florence Proux

Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time. DOI: http://dx.doi.org/10.7554/eLife.02230.001

Transcription Factors Modulate c-Fos Transcriptional Bursts

SUMMARY Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)—especially in rapidly inducible systems—is largely unknown. Using fluorescence in situ hybridization and computational models, we quantified the transcriptional activity of the proto-oncogene c-Fos with single mRNA accuracy at individual endogenous alleles. We showed that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with TALE DNA-binding domains, we systematically altered different aspects of these bursts. Specifically, we linked the polymerase initiation frequency to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. Our results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes.

A mutation in T7 RNA polymerase that facilitates promoter clearance

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo

DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5′-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.

Single cell correlation fractal dimension of chromatin: a framework to interpret 3D single molecule super-resolution.

Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications.

Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly.

DEAD‐box RNA‐dependent ATPases are ubiquitous enzymes that participate in nearly all processes involving RNA, but their detailed molecular functions remain generally unknown. SrmB, one of the five Escherichia coli DEAD‐box proteins, participates in the assembly of the large ribosomal subunit notably by facilitating the incorporation of L13, one of the ribosomal proteins that bind 23S rRNA earliest. Previously, we showed that SrmB is tethered to nascent ribosome through interactions with L4, L24 and the region from domain I of 23S rRNA that binds them. To identify the sites of action of SrmB, we have characterized rRNA mutations that bypass SrmB requirement. Five of them affect the same position from two repeated heptanucleotides in domain II of 23S rRNA, whereas two others affect a complementary hexanucleotide in 5S rRNA. Thus the sites of action of SrmB differ from its tethering site. In the mature ribosome, one of the heptanucleotides participates in a highly compact structure that contacts L13, the ‘1024 G‐ribo wrench’. In addition, we have observed that the assembly defect of ΔsrmB cells worsens as rRNA synthesis increases. Based on these results, we propose two non‐exclusive scenarios for the role of SrmB in ribosome assembly.

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

Significance The positive transcription elongation factor (P-TEFb) is required for transcription of most genes by RNA polymerase II. Hexim proteins associated to 7SK, a noncoding RNA, bind to P-TEFb and reversibly inhibit its activity. This transcription factor comprises the kinase Cdk9 and a cyclin T. Using a photoreactive amino acid incorporated into proteins, we provide the first evidence of a direct interaction between Cdk9 and Hexim1, in live cells, in cell extracts and in in vitro assembled complexes. An evolutionary conserved Hexim1 peptide, the “PYNT” sequence, cross-links to the Cdk9 activation segment that controls access to the catalytic cleft. Interference with binding of substrates accounts for kinase inhibition. The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide (202PYNTTQFLM210) known as the “PYNT” sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate binding-site. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.

The Deleterious Effect of an Insertion Sequence Removing the Last Twenty Percent of the Essential Escherichia coli rpsA Gene Is Due to mRNA Destabilization, Not Protein Truncation

Ribosomal protein S1, the product of the essential rpsA gene, consists of six imperfect repeats of the same motif. Besides playing a critical role in translation initiation on most mRNAs, S1 also specifically autoregulates the translation of its own messenger. ssyF29 is a viable rpsA allele that carries an IS10R insertion within the coding sequence, resulting in a protein lacking the last motif (S1DeltaC). The growth of ssyF29 cells is slower than that of wild-type cells. Moreover, translation of a reporter rpsA-lacZ fusion is specifically stimulated, suggesting that the last motif is necessary for autoregulation. However, in ssyF29 cells the rpsA mRNA is also strongly destabilized; this destabilization, by causing S1DeltaC shortage, might also explain the observed slow-growth and autoregulation defect. To fix this ambiguity, we have introduced an early stop codon in the rpsA chromosomal gene, resulting in the synthesis of the S1DeltaC protein without an IS10R insertion (rpsADeltaC allele). rpsADeltaC cells grow much faster than their ssyF29 counterparts; moreover, in these cells S1 autoregulation and mRNA stability are normal. In vitro, the S1DeltaC protein binds mRNAs (including its own) almost as avidly as wild-type S1. These results demonstrate that the last S1 motif is dispensable for translation and autoregulation: the defects seen with ssyF29 cells reflect an IS10R-mediated destabilization of the rpsA mRNA, probably due to facilitated exonucleolytic degradation.