Rhodobacter sphaeroides CarD Negatively Regulates Its Own Promoter

Bioinformatic analysis showed previously that a majority of promoters in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides lack the thymine at the last position of the -10 element (-7T), a base that is very highly conserved in promoters in bacteria other than α-proteobacteria. The absence of -7T was correlated with low promoter activity using purified R sphaeroides RNA polymerase (RNAP), but the transcription factor CarD compensated by activating almost all promoters lacking -7T tested in vitro , including rRNA promoters. Here we show that a previously uncharacterized R. sphaeroides promoter, the promoter for carD itself, has high basal activity relative to other tested R. sphaeroides promoters despite lacking -7T, and its activity is inhibited rather than activated by CarD. This high basal activity is dependent on a consensus extended -10 element (TGn) and specific features in the spacer immediately upstream of the extended -10 element. CarD negatively autoregulates its own promoter by producing abortive transcripts, limiting promoter escape and reducing full length mRNA synthesis. This mechanism of negative regulation differs from that employed by classical repressors, in which the transcription factor competes with RNA polymerase for binding to the promoter, and with the mechanism of negative regulation used by transcription factors like DksA/ppGpp and TraR that allosterically inhibit the rate of open complex formation. IMPORTANCE R. sphaeroides CarD activates many promoters by binding directly to RNAP and to DNA just upstream of the -10 element. In contrast, we show here that CarD inhibits its own promoter using the same interactions with RNAP and DNA used for activation. Inhibition results from increasing abortive transcript formation, thereby decreasing promoter escape and full-length RNA synthesis. We propose that the combined interactions of RNAP with CarD, with the extended -10 element, and with features in the adjacent -10/-35 spacer DNA stabilize the promoter complex, reducing promoter clearance. These findings support previous predictions that the effects of CarD on transcription can be either positive or negative, depending on the kinetic properties of the specific promoter.

Structural visualization of de novo initiation of RNA polymerase II transcription

Structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription were previously facilitated by the use of synthetic oligonucleotides. Here we report structures of initiation complexes de novo converted from pre-initiation complex (PIC) through catalytic activities and stalled at different template positions. Contrary to previous models, the closed-to-open promoter transition was accompanied by a large positional change of the general transcription factor TFIIH which became in closer proximity to TFIIE for the active delivery of the downstream DNA to the pol II active center. The initially-transcribing complex (ITC) reeled over 80 base pairs of the downstream DNA by scrunching, while retaining the fixed upstream contact, and underwent the transition to elongation when it encountered promoter-proximal pol II from a preceding round of transcription. TFIIH is therefore conducive to promoter melting, TSS scanning, and promoter escape, extending far beyond synthesis of a short transcript.

Structural basis of the complete poxvirus transcription initiation process

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I likely fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 Å. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

Structural basis of ribosomal RNA transcription regulation

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

Structural basis for transcription inhibition by E. coli SspA

Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.