Guiding strand passage: DNA-induced movement of the gyrase C-terminal domains defines an early step in the supercoiling cycle

DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA in a strand passage mechanism. A double-stranded segment of DNA, the T-segment, is passed through the gap in a transiently cleaved G-segment by coordinated closing and opening of three protein interfaces in gyrase. T-segment capture is thought to be guided by the C-terminal domains of the GyrA subunit of gyrase that wrap DNA around their perimeter and cause a DNA-crossing with a positive handedness. We show here that the C-terminal domains are in a downward-facing orientation in the absence of DNA, but swing up and rotate away from the gyrase body when DNA binds. The upward movement of the C-terminal domains is an early event in the catalytic cycle of gyrase that is triggered by binding of a G-segment, and first contacts of the DNA with the C-terminal domains, and contributes to T-segment capture and subsequent strand passag

A novel dimerization motif in the C-terminal domain of the Thermus thermophilus DEAD box helicase Hera confers substantial flexibility

DEAD box helicases are involved in nearly all aspects of RNA metabolism. They share a common helicase core, and may comprise additional domains that contribute to RNA binding. The Thermus thermophilus helicase Hera is the first dimeric DEAD box helicase. Crystal structures of Hera fragments reveal a bipartite C-terminal domain with a novel dimerization motif and an RNA-binding module. We provide a first glimpse on the additional RNA-binding module outside the Hera helicase core. The dimerization and RNA-binding domains are connected to the C-terminal RecA domain by a hinge region that confers exceptional flexibility onto the helicase, allowing for different juxtapositions of the RecA-domains in the dimer. Combination of the previously determined N-terminal Hera structure with the C-terminal Hera structures allows generation of a model for the entire Hera dimer, where two helicase cores can work in conjunction on large RNA substrate

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrat

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone

eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A's regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a ‘half-open' conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the ‘half-open' conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the ‘half-open' and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiatio

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

DEAD-box proteins disrupt or remodel RNA and protein/RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo- and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in non-canonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the P-loop and other helicase signature motif

Tiefencharakterisierung des intestinalen Transkriptoms der Maus mittels RNA-Seq

Die Schleimhaut des Darmtraktes ist charakterisiert durch komplexe metabolische und immunologische Prozesse und wird gesteuert durch hochdynamische Genexpressions-Programme. Mit der Verfügbarkeit von next generation sequencing und ihrer Nutzbarkeit für die Analyse von RNA-Sequenzen wurde die Genauigkeit über die globale Architektur des Transkriptoms gegenüber anderen Methoden wie microarrays auf eine neue Ebene befördert. Basierend auf dem 3‘ Oligo-dT priming von polyadenylierten Boten-RNA gefolgt von reverser Transkription und einem sogenannten template switch wurde eine Methode für RNA-Seq auf der Life Technologies SOLiD Plattform etabliert. Dieser Ansatz zeigte zuverlässige Informationen über das untersuchte Gewebe, z. B. in Bezug auf Genexpression und der Beobachtung von nicht-annotierten, transkriptionell aktiven Bereichen im Genom. In dieser Arbeit wird über die Tiefencharakterisierung des polyadenylierten Transkriptoms in zwei nah verwandten, dennoch unterschiedlichen Geweben des murinen Intestinaltrakts (Dünn- und Dickdarm) berichtet. Eine gewebespezifische Architektur des Transkriptoms und die Präsenz von zuvor nicht bekannten Bereichen transkriptioneller Aktivität wurden gefunden. Im ersten Schritt wurden Signaturen von 20.541 NCBI-RefSeq-Transkripten im Darm identifiziert (74,1 % der annotierten Gene), davon fanden sich 16.742 in beiden untersuchten Geweben. Obwohl die Mehrheit der Sequenzen annotierten Genen zugeordnet werden konnten, fanden sich 27.543 nicht-annotierte, transkriptionell aktive Regionen im Genom im Widerspruch zur aktuellen Genannotationen von RefSeq oder Ensembl. Unter Nutzung eines zweiten, unabhängigen, strangspezifischen Protokolls konnten 20.966 dieser Befunde bestätigt werden, die Mehrheit davon in direkter Nähe zu bekannten Genen. In der Folge wurden die Befunde bezüglich ihrer Nähe zu beschriebenen Exon-Elementen kategorisiert. Regionale Unterschiede zwischen Dünn- und Dickdarm dieser transkriptionell-aktiven, aber nicht annotierten Elemente wurden untersucht. Die vorliegende Arbeit demonstriert die Komplexität eines typischen intestinalen mRNA-Transkriptoms von Säugetieren anhand einer strangspezifischen Auflösung bis hin zur Einzelbase. Die Analysen zeigten zum ersten Mal ein strangspezifisches Bild von nicht-annotierten, transkriptionell aktiven Bereichen in zwei Geweben und repräsentieren eine Ressource für weitere Untersuchungen transkriptioneller Prozesse, welche zur molekularen Gewebeidentität beitragen. Die mittels RNA-Seq in dieser Arbeit erhobenen Daten waren von hoher, zuvor nicht zugänglicher Qualität, so dass RNA-Seq in der Zukunft vermutlich die neue Standardmethode für die genomweite Untersuchung des Transkriptoms darstellen wird.The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to other methods like microarrays. A method for RNA-Seq based on 3’ oligo-dT priming of polyadenylated messenger RNA followed by reverse transcription and a template switch was established on the Life Technologies SOLiD platform. This approach showed robust information about the characterized tissue, for example in terms of gene expression and the observation of non-annotated transcriptionally active regions of the genome. This thesis reports the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). The tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions were assessed. In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 non-annotated transcriptionally active regions not consistent with current gene annotations in RefSeq or Ensembl were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. This thesis further categorized the findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon. The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. The analysis for the first time shows a strand-specific comparative picture of non-annotated transcriptionally active regions in two tissues and represents a resource for further investigation of the transcriptional processes that contribute to molecular tissue identity. RNA-Seq generated data showed high, to date unseen quality. This suggests that RNA-Seq will be the new standard method for genome-wide analysis of the transcriptome

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domai