Protein–nucleic acid interactions: some assembly required

Protein–nucleic acid interactions: some assembly required Editorial overview James M Berger and Christoph W Mu¨ller Current Opinion in Structural Biology 2007, 17:77–79 Available online 23rd January 2007 0959-440X/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.sbi.2007.01.010

James M Berger QB3 Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3220, USA e-mail: [email protected]

James’ research employs structural and biochemical approaches to understand how ATP-dependent macromolecular assemblies control essential nucleic acid transactions in the cell. Primary areas of interest include the initiation of DNA replication, duplex unwinding and translocation events, and the maintenance of chromosome topology and superstructure. Christoph W Mu¨ller European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble Cedex 9, France e-mail: [email protected]

Christoph’s research addresses questions related to eukaryotic transcriptional regulation and chromatin structure. To gain insight, Christoph uses crystallography and electron microscopy combined with biochemistry and other biophysical techniques.

Proteins, like nature, abhor a vacuum. It is becoming ever more clear that many enzymes don’t act in isolation, but rather require the company of myriad other factors to appropriately regulate, direct and augment function. As a further testament to this need to combine activities, evolution often selects for multidomain proteins and protein complexes that contain large numbers of modular, but coupled, catalytic and scaffolding elements. Such architectures foster intermolecular and intramolecular crosstalk, which is vital for overcoming problems inherent to co-localization, enhancing functionality and improving mechanistic efficiency. The survey of recent developments outlined in this section of Current Opinion in Structural Biology shows that, when it comes to facilitating nucleic acid transactions and regulating genome metabolism, cells routinely rely on multifunctional proteins and assemblies to variously cut, join, pull and push DNA and RNA segments around. A wide variety of different types of ATP-fueled motor proteins translocate along or unwind nucleic acid duplexes to promote essential processes such as replication, transcription, DNA repair, genome packaging and chromatin remodeling. The review by Seidel and Dekker covers the state of the art in single-molecule studies of these machines, and the ingenious approaches researchers have devised to look at duplex unwinding, force generation, torque and NTP-driven catalytic events. Using single- or double-stranded nucleic acid segments attached to micron-sized beads, and by stretching or rotating these chains via laser traps, magnetic fields or fluid flow, motorinduced mechanical deformations and changes in DNA or RNA length can be readily monitored. Advances in instrumentation further permit direct measurement of the rates and processivity of DNA looping, supercoiling and particle movement; when coupled with tricks to slow these enzymes down (such as lowering NTP concentrations) or to improve instrument sensitivity, single step sizes can be determined. These fascinating studies are revealing important insights into how cooperativity arises between subunits in higher order translocase assemblies and set the stage for studies of complex events, such as the motor-directed displacement of nucleosomes and barrier proteins. How motor proteins precisely couple ATP hydrolysis to mechanical motion, and the extent to which translocation mechanisms are shared by different motor families is a related area of intense investigation. The review by Hopfner and Michaelis looks at new structural and biophysical findings that are helping to reveal how nucleic acid motor proteins physically flex in response to nucleotide binding and turnover to effect DNA or RNA movement. Here again, single-molecule studies play a pivotal role in delineating step size, progression rates and catalytic process. When coupled with structural studies, however, new and critical features of motor mechanisms further come to light. Structural studies of hexameric helicases and the

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Current Opinion in Structural Biology 2007, 17:77–79

78 Protein–nucleic acid interactions

distantly related F1 ATPase have implicated a rotary mechanism for nucleotide consumption, in which ATP hydrolysis progresses in a cyclic fashion around a protein ring to drive nucleic acid strands through the center of the particle. How the motor domains move in response to ATP turnover and how these motions control substrate transit is not yet understood, and may even differ between different helicase/translocase families. Structural comparisons between hexameric helicases, which translocate on one nucleic acid strand, and doublestranded DNA translocases indicate that there are both fundamental differences and intriguing similarities between motor mechanisms. Moreover, some of these chemo-mechanical coupling relationships can be extended to monomeric SF1 and SF2 helicases, whereas others appear distinct to each class of motor. Clearly, more structures of protein–substrate complexes and catalytic intermediates are needed, as are data for many members of each motor class, to begin to put together a comprehensive view of helicase/translocase function.

recently has the machinery that controls this event come to light at the structural level. In the Par system, which controls partitioning of a variety of plasmids, crystallographic studies have shown how relatively simple helixturn-helix elements have been augmented by auxiliary dimerization and DNA-binding domains to create a protein that can homo-oligomerize to synapse together distal regions of a centromere. This event in turn co-opts bacterial architectural factors to form a higher order nucleoprotein ‘segrosome’ that is competent to interact with other components of the partitioning machinery to direct plasmid anchoring and movement. Breakthroughs in the actual mechanics of plasmid separation came from structures of Par-type ATPases, which not only turn out to be homologs of eukaryotic actin, but also have been shown to form filamentous tracks within the cell. How the ParB elements interact with the ParA ATPase to accurately couple centromeric sites to this molecular highway and ensure faithful partitioning remains to be determined — stay tuned.

The review by Deaconescu, Savery and Darst focuses on a particular monomeric SF2 helicase, the bacterial transcription repair coupling factor (TRCF). TRCF is a complex, ATP-driven DNA translocase comprising seven distinct domains that collaborate in an intricate manner to simultaneously remove a stalled RNA polymerase (RNAP) from a damaged DNA template and recruit the Uvr(A)BC repair excinuclease to the faulty DNA site. TRCF overcomes RNAP blocks by binding upstream of the DNA lesion and pushing the enzyme in the downstream direction, thereby facilitating dissociation of the polymerase from the transcribed DNA or promoting the resumption of transcription. As outlined in the review, the complete crystal structure of unliganded TCRF provides new insights into both polymerase pushing and damage repair. Interestingly, the N-terminal moiety of TRCF is structurally similar to UvrB, part of the Uvr(A)BC excinuclease complex, in particular the region of UvrB that interacts with UvrA. This congruence suggests that TCRF recruits UvrA via its UvrB-like domain. Although the interacting surface is occluded by the C-terminal moiety of TRCF, various data support large rearrangements of TRCF during its functional cycle, which would expose the occluded surface. At the same time, the SF2 translocase domains hint at how force might be exerted on both the DNA and RNA polymerase to drive movement. Future structures with substrates are sure to provide fascinating insights into the mechanism of this remarkable composite machine.

In the next review, Rappas, Bose and Zhang introduce yet another class of Walker-A motif containing ATPase. The authors summarize recent insights into the function of bacterial enhancer-binding proteins (bEBPs) involved in activating s54–RNAP complexes during bacterial transcription. bEBP proteins belong to the family of AAA+ proteins (ATPases associated with various cellular activities). Similar to eukaryotic enhancer proteins, they bind DNA upstream of the transcription start site, oligomerize upon activation and ultimately remodel the stably closed s54–RNAP holoenzyme into an open complex with melted DNA where transcription can occur. The review presents recent structures of different bEBP proteins. Taken together, these structures suggest a sequence of events whereby initial DNA-bound dimers oligomerize to form hexameric rings stabilized by nucleotide binding. Subsequent nucleotide hydrolysis engages two conserved bEBP loops in the interaction with s54, which ultimately leads to open complex formation, promoter melting and transcription initiation. DNA binding, oligomerization and the conformational changes that occur upon nucleotide binding in the central domain of bEBP proteins appear reasonably well understood. In contrast, how interactions between the loops of the central domain and s54–RNAP remodel the s54–RNAP–DNA complex remains an open question. As in the previous reviews, structures of higher order complexes, in this case comprising bEBP proteins, s54 and, eventually, RNAP and promoter DNA, remain the next challenge to gain a more complete understanding of the mechanism of these complex assemblies.

ATP is also used by proteins to control genome segregation. A classic example of this process is outlined by Schumacher with regard to plasmid segregation. Although the players that ensure newly replicated plasmids are nonrandomly directed into both daughter bacteria upon cell division have been known and studied for decades, only Current Opinion in Structural Biology 2007, 17:77–79

The review by Kovall takes us from prokaryotic enhancer-binding proteins to the even more complicated transcriptional activation processes in eukaryotes. The Notch pathway plays a central role in communication between www.sciencedirect.com

Editorial overview Berger and Mu¨ller 79

adjacent cells, and regulates cell fate during early embryonic development and in the adult organism. The importance of the pathway becomes obvious when things go awry: misregulation of the Notch pathway can cause a number of diseases, including multiple sclerosis, various hereditary diseases and cancer. Central players of this pathway are the Notch receptor, the ligand DSL, the transcription factor CSL and the transcriptional co-activator Mastermind. In the past few years, several structures of components of the Notch signaling pathway have been determined. These include ankyrin repeat regions of the intracellular part of Notch (NotchIC) from several species, the structure of the CSL transcription factor–DNA complex and, recently, two independently determined structures of the active transcription complex comprising CSL bound to NotchIC and Mastermind co-regulators from Caenorhabditis elegans or human. The review by Kovall summarizes these structures and places particular emphasis on the central role of the transcription factor CSL. Once CSL is bound to NotchIC, interactions with co-repressors such as SMRT/N-CoR, CIR and Hairless are released, and new interactions with the Mastermind co-activator are established. CSL therefore functions as the central switch of the Notch pathway, integrating incoming signals and switching from a repressing to an activating function. Understanding this switch at the molecular level bears the promise that small molecules could be used to control its function; we expect the recent complex structures to contribute to finding such compounds. Once genes have been transcribed, the resultant mRNAs must be appropriately processed to facilitate translation. These and other RNAs are also subject to degradative control as a means of controlling the homeostasis of information flow. All living cells thus expend considerable amounts of energy on RNA synthesis, processing and turnover, the subjects of the two last reviews. Ribonucleases determine the lifetime of RNA molecules and play central roles in RNA metabolism. The different mechanisms of RNA processing and turnover often involve a combination of internal RNA cleavage and exonucleolytic cleavage at the 50 or 30 ends of the RNA. In the review by Worrall and Luisi, an overview of recently determined ribonuclease structures is presented, summarizing the latest findings on different endo- and exo-ribonucleases, including RNase E, RNase R and RNase II, RNase H, RNase III and the exosome. Enzymes that process and degrade RNA are ubiquitous, and the authors group different ribonucleases in the context of the Escherichia coli enzymes. Extracting general information from a wide range of different enzymes with different substrate specificities and related but distinct catalytic mechanisms is not an easy task. Nevertheless, a number of common principles emerge from this comparison. For example, it is noteworthy that multienzyme assemblies often combine different ribonuclease activities, as is the case for the bacterial degradosome and the www.sciencedirect.com

archaeal and eukaryotic exosomes. Coordination of enzymatic activities and processivity, and improved control over substrate access are probably the advantages of these assemblies. How RNA-processing enzymes tune their specific degrading functionalities and which structural features of RNA substrates help determine these different fates will hopefully become clearer with the availability of additional ribonuclease–substrate complexes. A particularly interesting family of ribonucleases that specifically cleave double-stranded RNA serves as the topic of the review by MacRae and Doudna. The RNase III group of RNA-processing enzymes currently attracts broad attention, because two family members, Dicer and Drosha, are responsible for processing RNA transcripts into microRNA (miRNAs) and short interfering RNAs (siRNAs). RNase III proteins are often multifunctional or multisubunit assemblies, and can be classified based on domain composition. Class I RNase III enzymes function as dimers, in which the RNase domains also act as dimerization domains, whereas class II and III family members are monomeric, forming a functional RNase from the internal fusion of two class I RNase III monomers. Comparing RNase III enzymes across a wide range of species leads the authors to conclude that RNase III enzymes use accessory domains as determinants of substrate specificity. For Dicer and Drosha, these accessory domains are the PAZ domain and the additional DGCR8 protein, respectively. Substrate specificity and catalytic domains are spatially separated and, in some instances, it appears that the RNase can precisely measure the distance between the RNA recognition and cleavage sites by using an internal scaffold element that functions as a molecular ruler. Given the number of different types of small RNAs and their importance in gene regulation and other cellular processes, there are sure to be many fundamental insights that will arise from the continued study of this essential protein family. In sum, although there may be a limited number of structural folds available in the protein universe, nature appears nearly boundless in her ability to evolve combinatorial arrays of domains and subunits that work together to catalyze critical nucleic acid transactions. As structural imaging techniques improve, the complexities of the large assemblies that run the cell, and the subtle macromolecular interactions by which these machines manipulate and process substrates will be revealed at atomicscale resolution. As single-molecule and solution-based methods of interrogation become more sensitive and sophisticated, key insights into the interplay between catalytic mechanism, nanoscale forces and dynamics will be uncovered. The integration of results from complementary experimental approaches into unifying and testable models will be essential to obtain a satisfactory description of large protein–nucleic acid assemblies, which are far more than the sum of their parts. Current Opinion in Structural Biology 2007, 17:77–79