Daniel Herschlag
Web: http://cmgm.stanford.edu/biochem/herschlag
Our research centers around the critical feature of biological systems, enzymatic catalysis. We are taking this problem in two directions: toward deriving a fundamental understanding of the chemical and physical principles that underlie enzymatic catalysis and towardunderstanding how these principles are utilized to produce complex biological processes. Each direction is described in turn below.
Principles of Enzymatic Catalysis
The basic question we are asking about enzymatic catalysis is: What distinguishes biological catalysts from simple chemical catalysts, allowing both RNA and protein enzymes to achieve their tremendous rate enhancements and exquisite specificity? We are taking several approaches to address this question, using RNA enzymes, protein enzymes and model systems.
RNA enzymes We are studying catalysis by RNA enzymes, or ribozymes, to learn about this 'new' class of enzymes and to compare and contrast the behavior of RNA enzymes with that of protein enzymes. The differences between the RNA and protein enzymes highlight features that are distinct, helping us to better understand each of these classes of biological macromolecules. Our studies have led to understanding differences in the folding, structural integrity, and structural redundancy of RNA and proteins and have led to the proposal that there are cellular proteins that act as 'RNA chaperones' (see below).
On the other hand, the features of protein and RNA enzymes that are similar may represent aspects that are fundamental to biological catalysis. Indeed, these studies have suggested that RNA enzymes, like their protein counterparts, can use binding interactions remote from the site of bond transformation to facilitate that transformation. Beyond this, recent results suggest that RNA enzymes are ideally suited for exploration of the energetic origins of this interconnection between binding and catalysis. This use of binding energy provides a natural connection between rate enhancement and specificity, the two hallmarks of biological catalysis.
The specific RNA catalysts currently under investigation are the group I self-splicing intron from Tetrahymena thermophila and the hammerhead ribozyme.
Protein enzymes
We focus on protein catalysis of reactions of phosphoryl compounds, the most prevalent class of biological reactions. The wealth of information obtained for these enzymes over decades of study allows us to design experiments probing specific mechanistic proposals. For example, do enzymes alter a reaction's transition state? What are the interactions that are strengthened in the reaction's transition state and what features and properties of the active site are responsible? What is the role of enzymatic rigidity and motion in catalysis? Techniques to address these questions include pre-steady state kinetics, site-directed mutagenesis, x-ray crystallography and spectroscopy, which are combined with the approaches of physical organic chemistry to address these and other questions. Protein enzymes that have been investigated in the lab include E. coli alkaline phosphatase, ribonuclease, the ras GTPase and NDP kinase.
To model studies to understand how an enzyme catalyzes a reaction, we must first understand the reaction itself. We are probing the nature of the transition state for phosphoryl transfer in solution and in enzyme-catalyzed reactions. We must know what these transition states look like in order to understand what active site interactions can provide selective stabilization of the reaction's transition state. Another long-standing unanswered question we are probing is what are the features of phosphoanhydrides such as ATP and pyrophosphate that impart their high energy nature, which is exploited as the cellular energy currency.
An understanding of enzymatic catalysis also requires an understanding of the physical interactions present within active sites. For this reason we have embarked on a study of the energetics of hydrogen bonding and effect of environment on the equilibria for hydrogen bond formation. The results to date with model systems suggest that solvents that mimic the low effective dielectric environment of protein active sites allow larger increases in hydrogen bond strength, which could in turn contribute to catalysis. Many fundamental features of hydrogen bonds and hydrogen bonding remain to be understood, and this is an active area of investigation in the lab. In the future we hope to be able study analogous questions with proteins. We are also initiating an investigation to quantitatively assess the rate enhancements achievable by pre-positioning reactive groups, either by covalent connections within small molecules or by binding interactions in enzyme active sites.
Complex Biological Processes
The same physical and chemical principles that underlie enzymatic catalysis more broadly define and delineate the capabilities and limitations of all biological systems. We are exploring the molecular and mechanistic basis of several complex biological processes, both through collaborations and through work centered in our own lab.
The work ongoing in the lab centers around two areas:
- How RNA structure is 'dealt with' in vivo, and how do proteins facilitate RNA structural rearrangements?
- The molecular mechanism for translation initiation.
RNA Structural Rearrangements
RNA has a strong tendency, in vitro, to fold into inactive structures that are kinetically stable. We are studying the process of RNA folding using the Tetrahymena group I ribozyme. The goals are to understand the molecular features that determine the rate and stability of the correctly folded structure, that affect the partitioning between active and inactive structures, and that allow RNA to fold cooperatively. The Tetrahymena ribozyme is particularly amenable to such detailed analysis because of the wealth of previous studies performed with this RNA and because the reaction catalyzed by this RNA provides a powerful read-out for formation of the correct structure.
Continuing on the theme of RNA misfolding, we and others have suggested that the tendency to fold into kinetically stable, inactive structures is a basic property of RNA, in large part deriving from the low information content provided by only 4 bases and from the high stability of RNA secondary structure. This has led to the suggestion that cellular RNAs have this same problem and that it is addressed by proteins that act as RNA chaperones to prevent RNA misfolding and to resolve RNAs that have misfolded. We have and would like to continue studying RNA¥protein interactions pertinent to these rearrangements and we would like to understand the mechanism by which proteins rearrange RNA structures.
One important class of proteins that appear to be involved in rearranging RNA structure is the 'DEAD' box family of RNA-dependent ATPases. These proteins share 7 motifs (one of which has the sequence DEAD) and play a role in virtually all cellular processes that involve RNA: mRNA transport and localization, pre-mRNA splicing, ribosome assembly, translation initiation (see below) and RNA turnover. Yet in no case is the molecular action of the DEAD box protein understood. Because of homology to DNA helicases and because several family members have been shown to have modest ability to unwind RNA duplexes, these proteins have uniformly been referred to as 'RNA helicases'. But, as we and others have pointed out, long stretches of duplex RNA are rarely encountered in the cell so that a role for these proteins as a canonical, processive helicase is unlikely. Further, RNA appears to be typically associated with proteins in vivo, so that a potential role for the DEAD box proteins is to rearrange protein¥RNA interactions, in addition to rearranging RNA structures; it is also possible that the energy of ATP hydrolysis is used to translocate along an RNA or RNP. We are trying to understand what these proteins do to RNA structures and trying to understand, at the most fundamental level, how they couple the energy of ATP hydrolysis to the conformational work that carries out these structural rearrangements.
It has been suggested that RNA may be particularly suited for involvement in processes that require structural rearrangements, such as protein synthesis by the ribosome and pre-mRNA splicing by the snRNAs and associated proteins. Group I introns self-splice in two distinct chemical steps, and switching between these steps requires multiple conformational rearrangements. We are studying the nature, thermodynamics and kinetics of these rearrangements using the Tetrahymena group I intron. The rationale is that this system is complex enough to undergo interesting conformational rearrangements, but simple enough to be experimentally tractable (38).
Eukaryotic Translation Initiation
Translation is central to biology, and the control of translation typically occurs at the level of initiation. Yet we know very little, beyond the molecules involved, about how initiation sites are found and about the similarities and distinctions in this process from one RNA to another. For example, it has been suggested that eIF4A, a DEAD box protein, is involved in unwinding structured regions in the 5' untranslated regions (UTRs) of mRNAs. However, there is no direct experimental support for this model. Also, ~90% of all messages use the 5'-most AUG for initiation, yet the molecular basis for this choice is not known.
We have recently embarked on a three-pronged approach to unravel the mechanistic basis of eukaryotic translation initiation:
- We are reconstituting translation initiation and following the kinetics of individual complex formation along the path to initiation. It is hoped that this will allow us to incisively probe the role of individual factors by manipulating the concentrations of wild-type and mutant factors, in conjunction with changes in the mRNA template, and determining the effects on individual steps within the overall initiation process. These studies may also teach us how to isolate and subsequently study intermediates in the initiation process.
- We are studying the mechanism of individual initiation factors in detail, starting with eIF4A and eIF4B, which together can unwind RNA duplexes. We would like to understand the nature of this activity (see above), how it relates to the physiological role of these proteins, and how the functions of these factors are integrated within the eIF4F complex.
- We are utilizing genomics approaches to track all of the mRNAs in yeast. The general strategy here is to look at mRNA in high vs low molecular weight polysomes -i.e., mRNAs that are efficiently or inefficiently initiated, and to use DNA arrays (in collaboration with Pat Brown) to look simultaneously at all of the mRNAs. We then probe the effects of mutations in individual initiation factors on the inclusion of each mRNA in high vs low molecular weight polysomes. We expect that this approach will be a rich source of models to be tested by additional genetic approaches and by the approaches outlined in points i and ii above.
Herschlag, D. (1995) J. Biol. Chem. 270, 20871-20874. "RNA Chaperones and the RNA Enzyme for Catalysis Positioning and Substrate Destabilization." (Medline)
Maegely, K.A., Admiraal, S.J. and Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8160-8166. "Ras-catalyzed Hydrolysis of GTP: Insights from Model Studies." (Medline)
Shan, S. and Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14474-14479. "The Change in Hydrogen Bond Strength Accompanying Charge Rearrangement: Implications for Enzymatic Catalysis."
Narlikar, G.J. and Herschlag, D. (1997) Annu. Rev. Biochem. 66, 19-59. "Mechanistic Aspects of Enzymatic Catalysis: Lessons from Comparison of RNA and Protein Enzymes."
Lorsch, J.R. and Herschlag, D. (1998) Biochemistry 37, 2180-2193. "The DEAD Box Protein eIF-4A 1. "A Kinetic and Thermodynamic Framework Revelas Coupled Binding of RNA and Nucleotide."
Narlikar, G.J. and Herschlag, D. (1998) Biochemistry 37, 9902-9911. "Direct Demonstration of the Catalytic Role of Binding Interactions in Enzymatic Reactions."
Peracchi, A., Beigelman, L., Karpeisky, A., Maloney, L., and Herschlag, D. (1998) Biochemistry 37, 14765-14775. "A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for its Extraordinary Sensitivity to Abasic Mutations."
O'Brien, P. and Herschlag, D. (1998) "Sulfatase Activity of E. Coli Alkakine Phosphatase Demonstrates a Functional Link to Arylsulfatases, an Evolutionarily Related Enzyme Family." J. Am. Chem. Soc., In press.
