NTP Binding Research Articles

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

Absolute binding free energies of mucroporin and its analog mucroporin-M1 with the heptad repeat 1 domain and RNA-dependent RNA polymerase of SARS-CoV-2

The peptide Mucroporin and its analog Mucroporin-M1 were studied using the molecular docking and molecular dynamics simulation of their complexation with two protein targets, the Heptad Repeat 1 (HR1) domain and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. The molecular docking of the peptide-protein complexes was performed using the glowworm swarm optimization algorithm. The lowest energy poses were submitted to molecular dynamics simulation. Then, the binding free energies of Mucroporin and its analog Mucroporin-M1 with these two protein targets were calculated using the Multistate Bennett Acceptance Ratio (MBAR) method. It was verified that the peptides/HR1 domain complex showed stability in the interaction site determined by molecular docking. It was also found that Mucroporin-M1 has a much higher affinity than Mucroporin to the HR1 protein target. The peptides showed similar stability and affinity at the NTP binding site in the RdRp protein. Additional experimental studies are needed to confirm the antiviral activity of Mucroporin-M1 and a possible mechanism of action against SARS-CoV-2. However, here we indicate that Mucroporin-M1 may have potential antiviral activity against the HR1 domain with the possibility for further peptide optimization. Communicated by Ramaswamy H. Sarma

Obligate movements of an active site–linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.

The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2′OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2′dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2′dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2′OH group to promote NTP binding, but selectively inhibits incorporation of 2′dNTPs by interacting with their 3′OH group to favor the catalytically-inert 2′-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.

A comprehensive mechanism for 5-carboxylcytosine-induced transcriptional pausing revealed by Markov state models

RNA polymerase II (Pol II) surveils the genome, pausing as it encounters DNA lesions and base modifications and initiating signals for DNA repair among other important regulatory events. Recent work suggests that Pol II pauses at 5-carboxycytosine (5caC), an epigenetic modification of cytosine, because of a specific hydrogen bond between the carboxyl group of 5caC and a specific residue in fork loop 3 of Pol II. This hydrogen bond compromises productive NTP binding and slows down elongation. Apart from this specific interaction, the carboxyl group of 5caC can potentially interact with numerous charged residues in the cleft of Pol II. However, it is not clear how other interactions between Pol II and 5caC contribute to pausing. In this study, we use Markov state models (a type of kinetic network models) built from extensive molecular dynamics simulations to comprehensively study the impact of 5caC on Pol II translocation. We describe two translocation intermediates with specific interactions that prevent the template base from loading into the Pol II active site. In addition to the previously observed state with 5caC constrained by fork loop 3, we discovered a new intermediate state with a hydrogen bond between 5caC and fork loop 2. Surprisingly, we find that 5caC may curb translocation by suppressing kinking of the helix bordering the active site (the bridge helix) because its high flexibility is critical to translocation. Our work provides new insights into how epigenetic modifications of genomic DNA can modulate Pol II translocation, inducing pauses in transcription.

Known Inhibitors of RNA Helicases and Their Therapeutic Potential.

RNA helicases are proteins found in all kingdoms of life, and they are associated with all processes involving RNA from transcription to decay. They use NTP binding and hydrolysis to unwind duplexes, to remodel RNA structures and protein-RNA complexes, and to facilitate the unidirectional metabolism of biological processes. Viral, bacterial, and eukaryotic parasites have an intimate need for RNA helicases in their reproduction. Moreover, various disorders, like cancers, are often associated with a perturbation of the host's helicase activity. Thus, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. In this review, we provide an overview of the different targeting strategies against helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on the proteins, and the therapeutic potential of these compounds in the treatment of various disorders.

Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Crystal structure of the NS3 helicase of tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus in Flaviviridae. It can cause the server infectious diseases named tick-borne encephalitis (TBE), which is characterized by paralysis and epilepsy. However, no effective treatment for TBE has been developed targeting TBEV. The NS3 helicase from TBEV plays an essential role in viral replication, which makes it an important target for drug design. In this study, the crystal structure of TBEV NS3 helicase has been determined to the resolution of 2.14 Å. Subsequent alignment with homologous structures reveals that the NTP binding site and RNA-binding sites are located in motifs Ⅱ and Ⅵ of NS3 and the critical residues for binding are conserved across species in the genus, while the distinct conformation transition implies that the TBEV helicase need a different local rearrangement. This study demonstrates the key atomic-level features of TBEV helicase and provides basis for the design of antiviral drugs targeting TBEV helicase.

2′-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Monitoring Structural Transitions in RNA Polymerase using Single Molecule FRET

Structural transitions in protein machines play a central role in their function. Such motions can be studied in great detail and in real-time using the method of single-molecule FRET (smFRET); however, the use of smFRET in visualizing protein motions has been limited by a lack of strategies for site-specific labeling of mobile protein modules. To address this challenge, we have developed methods for site-specific introduction of fluorescent probes into proteins such as the bacterial RNA polymerase (RNAP); the methods are highly selective and combine unnatural-amino-acid mutagenesis and bio-orthogonal labeling. We combine this breakthrough with smFRET on surface-immobilized molecules and look at conformational change of a large structural domain in the RNAP, “β clamp”. For the first time, we show directly that the clamp is highly dynamic, visiting three conformations. We also show that some antibiotics that target RNAP function by locking clamp into specific conformations; further, we discovered that a major stress-response factor (ppGpp) functions by modulating clamp dynamics. We then employed a similar strategy to a more difficult target, a less than 45 amino acid motif, “trigger loop” (TL), which is very close to the active center of RNAP. We succeeded in producing a fully functional TL-labeled RNAP and used smFRET to show directly that binding of correct NTP at the active site induces TL closure and that the TL ensures substrate specificity. Finally, we show using a real-time assay that TL opens and closes in each nucleotide addition cycle. Our strategies are general and provide insight into functioning of large multi-subunit protein machines.

Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.

Substrate Binding Regulates Redox Signaling in Human DNA Primase.

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.

Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA.

The σ factor drives promoter recognition by bacterial RNA polymerase (RNAP) and is also essential for later steps of transcription initiation, including RNA priming and promoter escape. Conserved region 3.2 of the primary σ factor (‘σ finger’) directly contacts the template DNA strand in the open promoter complex and facilitates initiating NTP binding in the active center of RNAP. Ribosomal RNA promoters are responsible for most RNA synthesis during exponential growth but should be silenced during the stationary phase to save cell resources. In Escherichia coli, the silencing mainly results from the action of the secondary channel factor DksA, which together with ppGpp binds RNAP and dramatically decreases the stability of intrinsically unstable rRNA promoter complexes. We demonstrate that this switch depends on the σ finger that destabilizes RNAP–promoter interactions. Mutations in the σ finger moderately decrease initiating NTP binding but significantly increase promoter complex stability and reduce DksA affinity to the RNAP–rRNA promoter complex, thus making rRNA transcription less sensitive to DksA/ppGpp both in vitro and in vivo. Thus, destabilization of rRNA promoter complexes by the σ finger makes them a target for robust regulation by the stringent response factors under stress conditions.

A computational approach for predicting off-target toxicity of antiviral ribonucleoside analogues to mitochondrial RNA polymerase

In the development of antiviral drugs that target viral RNA-dependent RNA polymerases, off-target toxicity caused by the inhibition of the human mitochondrial RNA polymerase (POLRMT) is a major liability. Therefore, it is essential that all new ribonucleoside analogue drugs be accurately screened for POLRMT inhibition. A computational tool that can accurately predict NTP binding to POLRMT could assist in evaluating any potential toxicity and in designing possible salvaging strategies. Using the available crystal structure of POLRMT bound to an RNA transcript, here we created a model of POLRMT with an NTP molecule bound in the active site. Furthermore, we implemented a computational screening procedure that determines the relative binding free energy of an NTP analogue to POLRMT by free energy perturbation (FEP), i.e. a simulation in which the natural NTP molecule is slowly transformed into the analogue and back. In each direction, the transformation was performed over 40 ns of simulation on our IBM Blue Gene Q supercomputer. This procedure was validated across a panel of drugs for which experimental dissociation constants were available, showing that NTP relative binding free energies could be predicted to within 0.97 kcal/mol of the experimental values on average. These results demonstrate for the first time that free-energy simulation can be a useful tool for predicting binding affinities of NTP analogues to a polymerase. We expect that our model, together with similar models of viral polymerases, will be very useful in the screening and future design of NTP inhibitors of viral polymerases that have no mitochondrial toxicity.

RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA–DNA base pair at the upstream edge of the transcription bubble. DNA–DNA base pairing immediately upstream from the RNA–DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA–DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

Fluorescence Kinetic Studies of DNA Unwrapping in Transcription Initiation with NTP addition and in Open Complex Dissociation by high salt

λPR promoter DNA is bent and wrapped around E. coli RNA polymerase (RNAP) in the stable open complex (RPo), as demonstrated by Cy3‐Cy5 fluorescent energy transfer (FRET) between dyes attached 115 bp apart on the far‐upstream (−100) and downstream (+14) position of DNA. Here we report the use of FRET and PIFE (single‐dye fluorescence enhancements that indicate close contacts between RNAP and the far‐upstream or downstream DNA) to investigate unwrapping of upstream DNA after NTP addition to initiate transcription, form a short RNA‐DNA hybrid and escape from the promoter. We observe two kinetic phases of roughly equal amplitude in the decrease in FRET in transcription initiation. The first kinetic phase (0.1 s to 10 s) of DNA unbending/unwrapping appears slower than the rate of NTP binding and dinucleotide synthesis, indicating that this first phase of unbending/unwrapping occurs in the processes of translocation and hybrid extension. The second kinetic phase (at times > 10 s) corresponds to that of production of full‐length (14 bp) RNA transcripts, as determined in parallel single‐round transcription assays. These results indicate that the second phase of DNA unbending and unwrapping occurs in promoter escape. In addition, we report the kinetics of unwrapping in dissociation by salt‐upshifts. These experiments reveal that salt‐induced unwrapping occurs during the conversion of the stable open complex to the unstable open intermediate I2, prior to DNA closing.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new l-glutaminase from Streptomyces pratensis NRC 10: Gene identification, enzyme purification, and characterization

In the current study, the purified l-glutaminase from Streptomyces pratensis NRC10 (GenBank number KC857622) was characterized. Its molecular weight was estimated to be 46kDa and isoelectric point 7.4. Its Vmax was calculated to be 2.19U/mg/min, while Km was 0.175mM. The optimum pH and temperature were 9 and 45°C, respectively. It was thermostable at 45°C but thermally inactivated at 60°C after 50min. Moreover, its enzymatic activity was enhanced by K+ ions and inhibited by Mg2+, Cu2+, Ag+, Hg2+, Ni2+, Fe2+, Cr2, Na+, Ca2+, and EDTA. A PCR fragment of 1550bp of S. pratensis NRC10 l-glutaminase gene (glsA) was purified and its sequence was determined (GenBank number KJ567136). l-glutaminase from NRC10 was induced mainly by l-glutamic acid. Model 3-D structure was composed of two domains, the serine - dependent beta-lactamase dominant the small STAS domain (Sulphate Transporter and anti-sigma factor antagonist) which had probably functioned as a general NTP binding domain. The two domains are linked by a linker peptide (GLHLMRNPALPGST), but sequence alignment between salt-tolerant glutaminase and the obtained glutaminase showed 44.75% of identity and 57% of similarity. This enzyme appears to have a distinctive structure compared to the rest of glutaminase family, and seems to construct a new subgroup of glutaminase.

Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest.

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.

Mechanism of NTP Binding to the Active Site of T7 RNA Polymerase Revealed by Free-Energy Simulation

In genetic transcription, molecular dynamic details and energetics of NTP binding to the active site of RNA polymerase (RNAP) are poorly understood. In this article, we investigated the NTP binding process in T7 RNAP using all-atom MD simulation combined with the umbrella sampling technique. Based on our simulations, a two-step mechanism was proposed to explain NTP binding: first, substrate NTP in aqueous solution, which carries a magnesium ion, diffuses through a secondary channel of RNAP to attain a pore region, where it undergoes conformational changes to give a correct orientation; next, the NTP establishes initial basepairing contacts with the template nucleoside (TN). Our free-energy calculations suggest that both steps are spontaneous. This mechanism can easily explain the problem of NTP binding with different orientations. Moreover, it is found that the nascent NTP:TN basepair is fragile and easily broken by thermal disturbance. Therefore, we speculate that the fingers domain will be triggered to close, so as to create a steady environment for the next chemical step. The observations from the work provide valuable information for comprehensively understanding the mechanism of the basic step in genetic transcription.