Preinitiation Complex Formation Research Articles

Abstract A12: Role of eIF5-mimic protein 1 (5MP1) for translational control in cancer

Abstract In eukaryotic translation initiation, both the m7G-capped mRNA and the small ribosomal subunit (SSU) are activated through eukaryotic initiation factors bound to Met-tRNAIMet, which allows formation of the 48S ribosome pre-initiation complex (PIC) with the small subunit attached to the mRNA 5' end. The PIC subsequently scans for a start codon, upon which it forms the ribosome initiation complex, ready for the polypeptide elongation, with the large subunit joined together. eIF5 is a crucial component of the multi-initiation factor complex (MFC) involved in the SSU activation and subsequent start codon selection by the 48S complex through its GTPase activating function (GAP). Paradoxically, however, overexpression turns eIF5 into an inhibitor of translation and decreases the accuracy of translation initiation, thereby allowing non-AUG initiation. Translation regulatory protein termed eIF5-mimic protein (5MP) exists in nearly all the eukaryotes except in nematodes, yeasts and some protozoans. Humans encode two of its copies, 5MP1 and 5MP2. It interacts with eIF2 and eIF3, major MFC components, and induces translation of ATF4 mRNA with regulatory uORFs. ATF4 is a pro-oncogenic transcription factor whose expression endorses tumor survival during stress conditions (hypoxia and nutritional deprivation) encountered by cancer cells in their development and metastasis. Moreover, preliminary study using yeast as a model system suggests that the universal role of 5MP in eukaryotic translation initiation is to increase the accuracy of translation initiation through inhibiting mis-initiation caused by excessive amount of eIF5. Here we examined the role of 5MP1 and 5MP2 in cultured human cells and found that 5MP1 expression suppresses non-AUG initiation of a luciferease reporter gene that occurs normally at a low level or at a higher level due to eIF5 overexpression, depending on its ability to bind the PIC. Furthermore, ribosome profiling studies in human cells show that expression of 5MP1 by itself inhibits non-AUG initiation of endogenous genes, such as GUG-initiated NAT1/eif4g2 and CUG-initiated c-Myc. These results indicate that 5MP in general suppresses non-AUG initiation through competing with eIF5 that occurs free of the ribosome. In support of the role of 5MP in cancer progression, 5MP1 knockdown and 5MP2 knockdown reduce the tumorigenicity of fibrosarcoma and salivary mucoepidermoid carcinoma, respectively. To test the model that 5MP promotes tumorigenicity through enhancing ATF4 expression, we examined correlation between eIF5 or 5MP expression and that of ATF4 target genes, using cancer genomics database. 5MP2 and eIF5 approximately correlated with the same subset of ATF4 targets in certain cancer types, but 5MP1 correlated with more ATF4 targets in more cancer types. These results not only confirm that eIF5 and 5MP promote tumorigenesis through up-regulating ATF4, but also suggest that 5MP1 has a greater role in cancer by unknown mechanism. Related to this point, we found a good correlation between 5MP1 and c-Myc expression in most types of cancers, and moreover c-Myc appears to be physically associated with 5MP1 promoters. Furthermore, in breast cancer high levels of 5MP1 are associated with poor prognosis, whereas high eIF5 levels correlate with more positive clinical outcomes. These results together suggest that, although up-regulation of ATF4 by eIF5 or 5MP is important, 5MP1 specially may play a major role in c-Myc-driven tumors, wherein a wider variety of ATF4 target genes are activated through 5MP1-ATF4 axis. The contrasting roles of 5MP1 and eIF5 in patient prognosis and accurate initiation suggests that suppression of non-AUG initiation may favor carcinogenesis. Collectively, future studies are warranted to establish the role of 5MP1 in cancer. Citation Format: Chelsea Moore, Sarah Gillaspie, Ji Wan, Eric Aube, Abbey Anderson, Chingakham Singh, Michael Witcher, Ivan Topisirovic, Shu-Bing Qian, Katsura Asano. Role of eIF5-mimic protein 1 (5MP1) for translational control in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A12.

Understanding the Mechanisms of Human RNA Polymerase II Transcription using Single Molecule Fluorescence Colocalization

Transcription is the first step in cellular gene expression and a key regulatory point for determining how a cell responds to stimuli. In mammalian cells, transcription is initiated by the formation of a pre-initiation complex (PIC), an assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II), on promoter DNA. Our goal is to use single molecule approaches to address mechanisms of PIC formation and activity that have previously been unattainable using traditional biochemical approaches. We have developed a novel single molecule total internal reflection fluorescence (TIRF) microscopy assay that allows us to visualize human Pol II transcriptional activity on individual DNA templates. With this single molecule setup, we have performed experiments to address the pathways by which active pre-initiation complexes can form. Ultimately we found that Pol II and three GTFs can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA.We are also developing a single molecule system to monitor dynamic protein-protein interactions that facilitate activation of transcription. It has long been known that transcriptional activators interact with subunits of transcription factor IID (TFIID) to potentiate high levels of transcription, however, the biophysical parameters defining these interactions are almost entirely uncharacterized. We have developed a novel method to label transcriptional activators and monitor their interactions with transcription factor TFIID in real-time using single molecule fluorescence colocalization. We observe that the activation domains from different activators exhibit different kinetic parameters for interactions with TFIID. We are positioned to address previously unanswered questions regarding how important protein-protein interactions between TFIID and transcriptional activators impact gene expression.

The TAF10-containing TFIID and SAGA transcriptional complexes are dispensable for early somitogenesis in the mouse embryo.

During development, tightly regulated gene expression programs control cell fate and patterning. A key regulatory step in eukaryotic transcription is the assembly of the pre-initiation complex (PIC) at promoters. PIC assembly has mainly been studied in vitro, and little is known about its composition during development. In vitro data suggest that TFIID is the general transcription factor that nucleates PIC formation at promoters. Here we show that TAF10, a subunit of TFIID and of the transcriptional co-activator SAGA, is required for the assembly of these complexes in the mouse embryo. We performed Taf10 conditional deletions during mesoderm development and show that Taf10 loss in the presomitic mesoderm (PSM) does not prevent cyclic gene transcription or PSM segmental patterning, whereas lateral plate differentiation is profoundly altered. During this period, global mRNA levels are unchanged in the PSM, with only a minor subset of genes dysregulated. Together, our data strongly suggest that the TAF10-containing canonical TFIID and SAGA complexes are dispensable for early paraxial mesoderm development, arguing against the generic role in transcription proposed for these fully assembled holo-complexes.

EIF4B stimulates eIF4A ATPase and unwinding activities by direct interaction through its 7-repeats region

ABSTRACTEukaryotic translation initiation starts with binding of the eIF4F complex to the 5′-m7G cap of the mRNA. Recruitment of the 43S pre-initiation complex (PIC), formed by the 40S ribosomal subunit and other translation initiation factors, leads to formation of the 48S PIC that then scans the 5′-untranslated region (5′-UTR) toward the start codon. The eIF4F complex consists of eIF4E, the cap binding protein, eIF4A, a DEAD-box RNA helicase that is believed to unwind secondary structures in the 5′-UTR during scanning, and eIF4G, a scaffold protein that binds to both eIF4E and eIF4A. The ATPase and helicase activities of eIF4A are jointly stimulated by eIF4G and the translation initiation factor eIF4B. Yeast eIF4B mediates recruitment of the 43S PIC to the cap-bound eIF4F complex by interacting with the 40S subunit and possibly with eIF4A. However, a direct interaction between yeast eIF4A and eIF4B has not been demonstrated yet. Here we show that eIF4B binds to eIF4A in the presence of RNA and ADPNP, independent of the presence of eIF4G. A stretch of seven moderately conserved repeats, the r1-7 region, is responsible for complex formation, for modulation of the conformational energy landscape of eIF4A by eIF4B, and for stimulating the RNA-dependent ATPase- and ATP-dependent RNA unwinding activities of eIF4A. The isolated r1-7 region only slightly stimulates eIF4A conformational changes and activities, suggesting that communication of the repeats with other regions of eIF4B is required for full stimulation of eIF4A activity, for recruitment of the PIC to the mRNA and for translation initiation.

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.

PIC Activation through Functional Interplay between Mediator and TFIIH

The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process.

Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP.

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).

Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture.

Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.

Crystal Structure of Human General Transcription Factor TFIIE at Atomic Resolution

In eukaryotes, RNA polymerase II requires general transcription factors to initiate mRNA transcription. TFIIE subunits α and β form a heterodimer and recruit TFIIH to complete the assembly of the pre-initiation complex. Here, we have determined the crystal structure of human TFIIE at atomic resolution. The N-terminal half of TFIIEα forms an extended winged helix (WH) domain with an additional helix, followed by a zinc-finger domain. TFIIEβ contains the WH2 domain, followed by two coiled-coil helices intertwining with TFIIEα. We also showed that TFIIEα binds to TFIIEβ with nanomolar affinity using isothermal titration calorimetry. In addition, mutations on the residues involved in the interactions resulted in severe growth defects in yeast. Lack of the C-terminal region of yeast TFIIEβ causes a mild growth defect in vivo. These findings provide a structural basis for understanding the functional mechanisms of TFIIE in the context of pre-initiation complex formation and transcription initiation.

Structure of the initiation-competent RNA polymerase I and its implication for transcription.

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing

Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dye-labeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing.1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013).

Nanoscale Probing of the p53 Tumor Suppression Transcription Machinery

More than 50% of cancer patients harbor p53 mutations, highlighting the essential role of p53 in tumor suppression. In response to various stress signals, p53 targets the TFIID-mediated transcription machinery to stimulate expression of vast gene networks involved in diverse cellular pathways. Understanding how p53 turns on TFIID-mediated transcription initiation at the single molecule level will advance our knowledge of p53's tumor suppression activity.Despite 20 years of biochemical studies, it remains elusive how p53 recruits TFIID and other basal transcription factors (e.g. TFIIB and RNA Polymerase II) to facilitate pre-initiation complex formation on various target gene promoters. We hypothesize that p53 induces distinct DNA-binding conformations within basal factors to stimulate assembly culminated in transcription initiation. Therefore, to test our hypothesis, we employed an innovative integrated approach involving single particle cryo-electron microscopy (EM), single molecule fluoresce microscopy and biochemistry.Intriguingly, our studies demonstrated that p53 delivers and promotes stable binding of TFIID to DNA. Unexpectedly, the interaction of TFIID and DNA in turn causes p53 to quickly dissociate from the assembly and promoter. Furthermore, our work suggest that the direct association of p53 and TFIID is sufficient to recruit TFIID onto various target gene promoters. More significantly, p53 induces a common DNA-binding conformation of TFIID. We further identified a novel role of TFIID in anchoring core promoter DNA downstream of the transcription start site.Collectively, these findings indicate that p53, and potentially activators in general, serve as escorts to dynamically recruit and load the basal transcription machinery onto DNA. Importantly, p53 facilitates formation of common DNA-binding forms of basal factors. Overall, our studies are critical for understanding how eukaryotic transcription complexes initiate transcription on different types of protein-coding genes in response to various stress signals.

The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1–Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

Dry and wet approaches for genome-wide functional annotation of conventional and unconventional transcriptional activators

Transcription factors (TFs) are master gene products that regulate gene expression in response to a variety of stimuli. They interact with DNA in a sequence-specific manner using a variety of DNA-binding domain (DBD) modules. This allows to properly position their second domain, called “effector domain”, to directly or indirectly recruit positively or negatively acting co-regulators including chromatin modifiers, thus modulating preinitiation complex formation as well as transcription elongation. At variance with the DBDs, which are comprised of well-defined and easily recognizable DNA binding motifs, effector domains are usually much less conserved and thus considerably more difficult to predict. Also not so easy to identify are the DNA-binding sites of TFs, especially on a genome-wide basis and in the case of overlapping binding regions. Another emerging issue, with many potential regulatory implications, is that of so-called “moonlighting” transcription factors, i.e., proteins with an annotated function unrelated to transcription and lacking any recognizable DBD or effector domain, that play a role in gene regulation as their second job. Starting from bioinformatic and experimental high-throughput tools for an unbiased, genome-wide identification and functional characterization of TFs (especially transcriptional activators), we describe both established (and usually well affordable) as well as newly developed platforms for DNA-binding site identification. Selected combinations of these search tools, some of which rely on next-generation sequencing approaches, allow delineating the entire repertoire of TFs and unconventional regulators encoded by the any sequenced genome.

Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor.

Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

BackgroundIn the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation.ResultsUsing a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo.ConclusionsThese studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation.

Initiation of mRNA translation in bacteria: structural and dynamic aspects.

Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) containing initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.