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Patrick Cramer

Publications and source records attributed to Patrick Cramer.

At least 19 recordsLinked to original sources
PubMed2026

Oriented binding of transcription factors to nucleosomes remodels chromatin at human promoters.

Transcription factors (TFs) can access nucleosomes via five distinct modes: gyre-spanning, periodic-binding, dyad-binding, and end-binding modes as well as an oriented binding mode, where the TF binding motif shows orientational preference relative to the nucleosome. Here, we report the first structure of an oriented TF:nucleosome complex, where two ELF2 proteins bind to a double motif located at superhelical location +4, unwinding four helical turns of DNA from the nucleosome. We further show that unlike previously described pioneer factors, ELF2 is able to occupy all of its unmethylated, high-affinity double motifs in vivo. Motifs of ELF2 and another oriented nucleosome binder, YY1, are highly enriched downstream of transcription start sites (TSSs) of highly expressed genes, with the motifs oriented in such a way that the TSS becomes accessible upon TF binding. Our results suggest that oriented binding may be generally important for high transcriptional activity.

Nucleosomes↗
PubMed2006

Template misalignment in multisubunit RNA polymerases and transcription fidelity.

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

Base Sequence↗
PubMed2006

Structure and TBP binding of the Mediator head subcomplex Med8-Med18-Med20.

The Mediator head module stimulates basal RNA polymerase II (Pol II) transcription and enables transcriptional regulation. Here we show that the head subunits Med8, Med18 and Med20 form a subcomplex (Med8/18/20) with two submodules. The highly conserved N-terminal domain of Med8 forms one submodule that binds the TATA box-binding protein (TBP) in vitro and is essential in vivo. The second submodule consists of the C-terminal region of Med8 (Med8C), Med18 and Med20. X-ray analysis of this submodule reveals that Med18 and Med20 form related beta-barrel folds. A conserved putative protein-interaction face on the Med8C/18/20 submodule includes sites altered by srb mutations, which counteract defects resulting from Pol II truncation. Our results and published data support a positive role of the Med8/18/20 subcomplex in initiation-complex formation and suggest that the Mediator head contains a multipartite TBP-binding site that can be modulated by transcriptional activators.

Amino Acid Sequence↗
PubMed2006

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

Amino Acid Sequence↗
PubMed2006

Fluorescence detection of nucleic acids and proteins in multi-component crystals.

A highly sensitive and specific assay for the rapid detection of nucleic acids in crystals of macromolecular complexes is reported. The assay is based on the observation of crystals under a fluorescence microscope after incubation with a fluorescent dye that specifically binds nucleic acids. The assay allows clear distinction between crystals of free RNA polymerase II and crystals of the polymerase bound to DNA and RNA or RNA alone, even when the nucleic acids account for only 3% of the crystalline material. Fluorescence detection was also used to monitor the diffusion and accumulation of a fluorescence-labelled polymerase-binding protein in preformed polymerase crystals. Additional experiments suggest the general applicability of these fluorescence assays for the characterization of crystal contents.

DNA↗
PubMed2006

Mechanistic studies of the mRNA transcription cycle.

We have now completed an atomic crystallographic model of the 12-subunit yeast RNA polymerase II in elongation mode, with DNA and RNA in the active-centre cleft, and the NTP substrate at the growing end of the RNA. From these studies has emerged a detailed three-dimensional view of mRNA elongation. We have extended this structural analysis to a polymerase elongation complex bound by the transcript cleavage factor TFIIS (transcription factor IIS), which is required for polymerase escape from DNA arrest sites. A detailed model of this complex reveals a single tuneable active site for RNA polymerization and cleavage, and changes in the position of the RNA and polymerase domains, reflecting the dynamic nature of the elongation complex. An additional structure of a polymerase CTD (C-terminal domain) phosphopeptide bound by the 3'-RNA processing factor Pcf11 provides insights into the coupling of transcription elongation to mRNA processing. The structure of the CTD phosphatase Scp1 trapped in an intermediary enzymatic state explains CTD dephosphorylation during recycling of the polymerase. We also recently reported the first crystal structure of a Mediator subcomplex, which reveals an extended helical fold with a conserved hinge.

Crystallography, X-Ray↗
PubMed2005

Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.

The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Base Sequence↗
PubMed2005

Structure and carboxyl-terminal domain (CTD) binding of the Set2 SRI domain that couples histone H3 Lys36 methylation to transcription.

During mRNA elongation, the SRI domain of the histone H3 methyltransferase Set2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. The solution structure of the yeast Set2 SRI domain reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues, whereas a single CTD repeat is insufficient for binding. Residues that show strong chemical shift perturbations upon CTD binding cluster in two regions. Both CTD tyrosine side chains contact the SRI domain. One of the tyrosines binds in the region with the strongest chemical shift perturbations, formed by the two NH2-terminal helices. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in bacterial sigma factors (domain sigma2 in sigma70).

Amino Acid Sequence↗
PubMed2005

Structure of the mediator subunit cyclin C and its implications for CDK8 function.

Cyclin C binds the cyclin-dependent kinases CDK8 and CDK3, which regulate mRNA transcription and the cell cycle, respectively. The crystal structure of cyclin C reveals two canonical five-helix repeats and a specific N-terminal helix. In contrast to other cyclins, the N-terminal helix is short, mobile, and in an exposed position that allows for interactions with proteins other than the CDKs. A model of the CDK8/cyclin C pair reveals two regions in the interface with apparently distinct roles. A conserved region explains promiscuous binding of cyclin C to CDK8 and CDK3, and a non-conserved region may be responsible for discrimination of CDK8 against other CDKs involved in transcription. A conserved and cyclin C-specific surface groove may recruit substrates near the CDK8 active site. Activation of CDKs generally involves phosphorylation of a loop at a threonine residue. In CDK8, this loop is longer and the threonine is absent, suggesting an alternative mechanism of activation that we discuss based on a CDK8-cyclin C model.

Amino Acid Sequence↗
PubMed2005

A structural perspective of CTD function.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) integrates nuclear events by binding proteins involved in mRNA biogenesis. CTD-binding proteins recognize a specific CTD phosphorylation pattern, which changes during the transcription cycle, due to the action of CTD-modifying enzymes. Structural and functional studies of CTD-binding and -modifying proteins now reveal some of the mechanisms underlying CTD function. Proteins recognize CTD phosphorylation patterns either directly, by contacting phosphorylated residues, or indirectly, without contact to the phosphate. The catalytic mechanisms of CTD kinases and phosphatases are known, but the basis for CTD specificity of these enzymes remains to be understood.

Amino Acid Sequence↗
PubMed2005

A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer.

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. We present the 3.0-A crystal structure of a highly conserved part of the Mediator, the MED7.MED21 (Med7.Srb7) heterodimer. The structure is very extended, spanning one-third of the Mediator length and almost the diameter of Pol II. It shows a four-helix bundle domain and a coiled-coil protrusion connected by a flexible hinge. Four putative protein binding sites on the surface allow for assembly of the Mediator middle module and for binding of the conserved subunit MED6, which is shown to bridge to the Mediator head module. A flexible MED6 bridge and the MED7.MED21 hinge could account for changes in overall Mediator structure upon binding to Pol II or activators. Our results support the idea that transcription regulation involves conformational changes within the general machinery.

Amino Acid Sequence↗
PubMed2005

The dynamic machinery of mRNA elongation.

Two complementary X-ray studies of the interaction between RNA polymerase II and nucleic acids have improved our understanding of mRNA elongation. These studies suggest how RNA polymerase II unwinds DNA, how it separates the RNA product from the DNA template and how it incorporates nucleoside triphosphate (NTP) substrates into the growing RNA chain. The tunable polymerase active center apparently allows repositioning of a catalytic metal ion, rotation of NTPs before their incorporation, RNA repositioning by a transcript cleavage factor, and modulation of enzyme activity by a bacterial small molecule regulator and its protein cofactor.

Catalysis↗
PubMed2005

An atypical spinal meningioma with CSF metastasis: fatal progression despite aggressive treatment. Case report.

The authors report the case of a 23-year-old man who presented with a C1-3 spinal mass. Following intraspinal decompression the tumor was histologically classified as an atypical meningioma (World Health Organization grade II). Two further surgical interventions resulted in almost total removal of the meningioma. In addition, radiotherapy was performed. During the 1.5-year follow-up period the diagnostic examinations identified a local tumor recurrence, an intraspinal C-6 metastasis, and a segmental instability with anterior C2-3 slippage and C3-4 kyphosis. The tumor was resected and occipitocervical stabilization was performed. Histological examination showed no change in malignancy. Despite additional hydroxyurea-based chemotherapy, the patient presented 4 months later with a hemiparesis and a massive recurrence of the tumor mass involving the posterior fossa and the upper thoracic spine. Because there were no further therapeutical options, the patient died. The authors discuss more aggressive therapeutic options in addition to surgery in patients with metastatic atypical meningiomas. The results in the reported case indicate that meningiomas associated with cerebrospinal fluid metastasis may represent a higher grade of malignancy.

Adult↗
PubMed2004

Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS.

The crystal structure of the complete 12 subunit RNA polymerase (pol) II bound to a transcription bubble and product RNA reveals incoming template and nontemplate DNA, a seven base pair DNA/RNA hybrid, and three nucleotides each of separating DNA and RNA. The complex adopts the posttranslocation state and accommodates a cocrystallized nucleoside triphosphate (NTP) substrate. The NTP binds in the active site pore at a position to interact with a DNA template base. Residues surrounding the NTP are conserved in all cellular RNA polymerases, suggesting a universal mechanism of NTP selection and incorporation. DNA-DNA and DNA-RNA strand separation may be explained by pol II-induced duplex distortions. Four protein loops partition the active center cleft, contribute to embedding the hybrid, prevent strand reassociation, and create an RNA exit tunnel. Binding of the elongation factor TFIIS realigns RNA in the active center, possibly converting the elongation complex to an alternative state less prone to stalling.

Amino Acid Sequence↗
PubMed2004

Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.

We determined the x-ray structure of the RNA polymerase (Pol) II subcomplex Rpb4/7 at 2.3 A resolution, combined it with a previous structure of the 10-subunit polymerase core, and refined an atomic model of the complete 12-subunit Pol II at 3.8-A resolution. Comparison of the complete Pol II structure with structures of the Pol II core and free Rpb4/7 shows that the core-Rpb4/7 interaction goes along with formation of an alpha-helix in the linker region of the largest Pol II subunit and with folding of the conserved Rpb7 tip loop. Details of the core-Rpb4/7 interface explain facilitated Rpb4/7 dissociation in a temperature-sensitive Pol II mutant and specific assembly of Pol I with its Rpb4/7 counterpart, A43/14. The refined atomic model of Pol II serves as the new reference structure for analysis of the transcription mechanism and enables structure solution of complexes of the complete enzyme with additional factors and nucleic acids by molecular replacement.

Amino Acid Sequence↗
PubMed2004

Structure and mechanism of RNA polymerase II CTD phosphatases.

Recycling of RNA polymerase II (Pol II) after transcription requires dephosphorylation of the polymerase C-terminal domain (CTD) by the phosphatase Fcp1. We report the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to those of phosphotransferases and phosphohydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. We demonstrate that the first aspartate in the signature motif undergoes metal-assisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for binding of Fcp1 to the polymerase in vitro.

Amino Acid Sequence↗