Evaluating elastic network models of crystalline biological molecules with temperature factors, correlated motions, and diffuse x-ray scattering

In this study, the variance-covariance matrix of protein motions is used to compare several elastic network models within the theoretical framework of x-ray scattering from crystals. A set of 33 ultra-high resolution structures is used to characterize the average scaling behavior of the vibrational density of states and make comparisons between experimental and theoretical temperature factors. Detailed investigations of the vibrational density of states, correlations, and predicted diffuse x-ray scatter are carried out for crystalline Staphylococcal nuclease; correlations and diffuse x-ray scatter are also compared to predictions from the translation, libration, screw model and a liquid-like dynamics model. We show that elastic network models developed to best predict temperature factors without regard for the crystal environment have relatively strong long-range interactions that yield very short-ranged atom-atom correlations. Further, we find that the low-frequency modes dominate the variance-covariance matrix only for those models with a physically reasonable vibrational density of states, and the fraction of modes required to converge the correlations is higher than that typically used for elastic network model studies. The practical implications are explored using computed diffuse x-ray scatter, which can be measured experimentally.     

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.     

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

   

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.     

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.     

Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways

Living cells possess a panel of specialized DNA polymerases that deal with the large diversity of DNA lesions that occur in their genomes. How specialized DNA polymerases gain access to the replication intermediate in the vicinity of the lesion is unknown. Using a model system in which a single replication blocking lesion can be bypassed concurrently by two pathways that leave distinct molecular signatures, we analyzed the complex interplay among replicative and specialized DNA polymerases. The system involves a single N-2-acetylaminofluorene guanine adduct within the NarI frameshift hot spot that can be bypassed concurrently by Pol II or Pol V, yielding a −2 frameshift or an error-free bypass product, respectively. Reconstitution of the two pathways using purified DNA polymerases Pol III, Pol II and Pol V and a set of essential accessory factors was achieved under conditions that recapitulate the known in vivo requirements. With this approach, we have identified the key replication intermediates that are used preferentially by Pol II and Pol V, respectively. Using single-hit conditions, we show that the β-clamp is critical by increasing the processivity of Pol II during elongation of the slipped −2 frameshift intermediate by one nucleotide which, surprisingly, is enough to support subsequent elongation by Pol III rather than degradation. Finally, the proofreading activity of the replicative polymerase prevents the formation of a Pol II-mediated −1 frameshift product. In conclusion, failure or success of TLS pathways appears to be the net result of a complex interplay among DNA polymerases and accessory factors.     

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).     

The two-step model for translesion synthesis: then and now

The formation of base substitution mutations following exposure of bacteria to ultraviolet light and many other mutagens occurs during translesion synthesis opposite a photoproduct or other lesion in the template strand of DNA. This process requires the UmuD(2)' UmuC complex, only formed to a significant extent in SOS-induced cells. The "two-step" model proposed that there were two steps, insertion of a wrong base (misincorporation) and use of the misincorporated base as a primer for further chain extension (bypass). The original evidence suggested that UmuD(2)' UmuC was needed only for the second step and that in its absence other polymerases such as DNA polymerase III could make misincorporations. Now we know that the UmuD(2)' UmuC complex is DNA polymerase V and that it can carry out both steps in vitro and probably does both in vivo in wild-type cells. Even so, DNA polymerase III clearly has an important accessory role in vitro and a possibly essential role in vivo, the precise nature of which is not clear. DNA polymerases II and IV are also up-regulated in SOS-induced cells and their involvement in the broader picture of translesion synthesis is only now beginning to emerge. It is suggested that we need to think of the chromosomal replication factory as a structure through which the DNA passes and within which as many as five DNA polymerases may need to act. Protein-protein interactions may result in a cassette system in which the most appropriate polymerase can be engaged with the DNA at any given time. The original two-step model was very specific, and thus an oversimplification. As a general concept, however, it reflects reality and has been demonstrated in experiments with eukaryotic DNA polymerases in vitro.     

A multibody modelling approach to determine load sharing between passive elements of the lumbar spine

The human spinal segment is an inherently complex structure, a combination of flexible and semi-rigid articulating elements stabilised by seven principal ligaments. An understanding of how mechanical loading is shared among these passive elements of the segment is required to estimate tissue failure stresses. A 3D rigid body model of the complete lumbar spine has been developed to facilitate the prediction of load sharing across the passive elements. In contrast to previous multibody models, this model includes a non-linear, six degrees of freedom intervertebral disc, facet bony articulations and all spinal ligaments. Predictions of segmental kinematics and facet joint forces, in response to pure moment loading (flexion-extension), were compared to published in vitro data. On inclusion of detailed representation of the disc and facets, the multibody model fully captures the non-linear flexibility response of the spinal segment, i.e. coupled motions and a mobile instantaneous centre of rotation. Predicted facet joint forces corresponded well with reported values. For the loading case considered, the model predicted that the ligaments are the main stabilising elements within the physiological motion range; however, the disc resists a greater proportion of the applied load as the spine is fully flexed. In extension, the facets and capsular ligaments provide the principal resistance. Overall patterns of load distribution to the spinal ligaments are in agreement with previous predictions; however, the current model highlights the important role of the intraspinous ligament in flexion and the potentially high risk of failure. Several important refinements to the multibody modelling of the passive elements of the spine have been described, and such an enhanced passive model can be easily integrated into a full musculoskeletal model for the prediction of spinal loading for a variety of daily activities.     

Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.     

A multibody modelling approach to determine load sharing between passive elements of the lumbar spine

The human spinal segment is an inherently complex structure, a combination of flexible and semi-rigid articulating elements stabilised by seven principal ligaments. An understanding of how mechanical loading is shared among these passive elements of the segment is required to estimate tissue failure stresses. A 3D rigid body model of the complete lumbar spine has been developed to facilitate the prediction of load sharing across the passive elements. In contrast to previous multibody models, this model includes a non-linear, six degrees of freedom intervertebral disc, facet bony articulations and all spinal ligaments. Predictions of segmental kinematics and facet joint forces, in response to pure moment loading (flexion–extension), were compared to published in vitro data. On inclusion of detailed representation of the disc and facets, the multibody model fully captures the non-linear flexibility response of the spinal segment, i.e. coupled motions and a mobile instantaneous centre of rotation. Predicted facet joint forces corresponded well with reported values. For the loading case considered, the model predicted that the ligaments are the main stabilising elements within the physiological motion range; however, the disc resists a greater proportion of the applied load as the spine is fully flexed. In extension, the facets and capsular ligaments provide the principal resistance. Overall patterns of load distribution to the spinal ligaments are in agreement with previous predictions; however, the current model highlights the important role of the intraspinous ligament in flexion and the potentially high risk of failure. Several important refinements to the multibody modelling of the passive elements of the spine have been described, and such an enhanced passive model can be easily integrated into a full musculoskeletal model for the prediction of spinal loading for a variety of daily activities.     

In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.     

Unifying themes in DNA replication: reconciling single molecule kinetic studies with structural data on DNA polymerases

Structural data suggest that DNA polymerases, from at least three different families, employ common strategies for carrying out DNA replication. Universal features include a large conformational change in the enzyme-template complex and a conserved active-site geometry that imposes a sharp kink at the 5 end of the template strand. Recent single molecule experiments have shown that stretching the DNA template markedly alters the rate of DNA synthesis catalyzed by these motor enzymes. From these data, it was previously inferred that T7 DNA polymerase and two related enzymes convert two or four (depending on the enzyme) single-stranded (ss) template bases to double helix geometry in the polymerase active site during each catalytic cycle. We discuss structural data on related DNA polymerases, which suggest that only one (ss) template base is contracted to dsDNA geometry during the rate-limiting step of each replication cycle. Previous interpretations relied upon the global stretching curves for DNA polymers alone (with no reference to the enzyme or the structure of the transition state). In contrast, we present a structurally guided model that presumes the force dependence of the replication rate is governed chiefly by local interactions in the immediate vicinity of the enzyme s active site. Our analysis reconciles single molecule kinetic studies with structural data on DNA polymerases.     

What Is the Role of Motif D in the Nucleotide Incorporation Catalyzed by the RNA-dependent RNA Polymerase from Poliovirus?

Poliovirus (PV) is a well-characterized RNA virus, and the RNA-dependent RNA polymerase (RdRp) from PV (3D^pol) has been widely employed as an important model for understanding the structure-function relationships of RNA and DNA polymerases. Many experimental studies of the kinetics of nucleotide incorporation by RNA and DNA polymerases suggest that each nucleotide incorporation cycle basically consists of six sequential steps: (1) an incoming nucleotide binds to the polymerase-primer/template complex; (2) the ternary complex (nucleotide-polymerase-primer/template) undergoes a conformational change; (3) phosphoryl transfer occurs (the chemistry step); (4) a post-chemistry conformational change occurs; (5) pyrophosphate is released; (6) RNA or DNA translocation. Recently, the importance of structural motif D in nucleotide incorporation has been recognized, but the functions of motif D are less well explored so far. In this work, we used two computational techniques, molecular dynamics (MD) simulation and quantum mechanics (QM) method, to explore the roles of motif D in nucleotide incorporation catalyzed by PV 3D^pol. We discovered that the motif D, exhibiting high flexibility in either the presence or the absence of RNA primer/template, might facilitate the transportation of incoming nucleotide or outgoing pyrophosphate. We observed that the dynamic behavior of motif A, which should be essential to the polymerase function, was greatly affected by the motions of motif D. In the end, through QM calculations, we attempted to investigate the proton transfer in enzyme catalysis associated with a few amino acid residues of motifs F and D.     

细菌转录起始调控机制*

原核生物同一种群的每个细胞都是和外界环境直接接触的,它们主要通过开启或关闭某些基因的表达来适应环境条件。所以,环境因子往往是调控的效应因子,必须严格调控转录来确保细胞对环境改变做出有效且充分的反应。原核生物基因的表达受多种因素的调控,而对于大多数细菌来说,调控基因表达的关键步骤是启动子识别和RNA聚合酶启动转录。在细菌的细胞中,可以通过调节RNA聚合酶的活性以及改变RNA聚合酶对启动子的结合来优化基因的转录过程以适应不同环境变化。总结了目前已发现的参与细菌细胞转录调节的各类因子,从这些因子对启动子的作用、RNA聚合酶的作用以及两者的相互作用等方面阐述它们调控基因表达的分子机制。总结多种基因调控的作用,加深对转录起始过程的认识,希望能对未来调控转录起始过程来实现目标基因的高效表达和不利基因的抑制表达提供思路,为以后的工业菌株改造提供依据。