We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

Single-molecule techniques have been developed for commercial DNA sequencing1,2. One emerging strategy uses a nanopore to analyze DNA molecules as they are driven electrophoretically in single file order past a sensor3-5. However, uncontrolled DNA strand electrophoresis through nanopores is too fast for accurate base reads6. A proposed solution would employ processive enzymes to deliver DNA through the pore at a slower average rate7. Here, we describe forward and reverse ratcheting of DNA templates through the α–hemolysin (α-HL) nanopore controlled by wild-type phi29 DNA polymerase (phi29 DNAP). DNA strands were examined in single file order at one nucleotide spatial precision in real time. The registry error probability (either an insertion or deletion during one pass along a template strand) ranged from 10% to 24.5% absent optimization. This general strategy facilitates multiple reads of individual template strands and is transferrable to other nanopore devices for implementation of DNA sequence analysis.

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3′-5′ exonuclease activity of wild-type phi29 DNAP to excise a 3′-H terminal residue, yielding a primer strand 3′-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Angstrom scale precision as the template strand was drawn through the nanopore lumen during replication.Single molecule techniques are now used routinely to study nucleic acids in basic science 1 -3 and technology 4 ,5 . Methods using nanoscale pores (nanopores) are advantageous because they can report the length, structure and composition of unmodified DNA or RNA molecules that are captured in single file order 6-9. Data are typically reported as a time series of ionic current as each DNA strand is driven by an applied electric field across a single pore controlled by a voltage-clamped amplifier. Hundreds to thousands of molecules can be examined at high bandwidth and spatial resolution.Recently, the properties of DNA or RNA molecules bound to nucleic acid processing enzymes have been analyzed at a nanopore orifice. The complexes studied include those of single-stranded DNA with Escherichia coli Exonuclease I 10 , RNA with the bacteriophage phi8 ATPase, 11 , and primer/template DNA substrates bound to the 3′-5′-exonuclease deficient versions of two A-family DNA polymerases, the Klenow fragment of E. coli DNA polymerase (KF(exo-)) and bacteriophage T7 DNA polymerase (T7DNAP(exo-)) 12 -16. We have demonstrated that T7DNAP(exo-) could replicate and advance a DNA template held in the α-hemolysin (α-HL) nanopore against an 80 mV applied potential 17. However, due to the low stability of the T7DNAP(exo-)-DNA complex under load, diminished signal * corresponding author: makeson@soe.ucsc.edu. Figure S1 (sequences of 5′-6-FAM, 3′-OH and 5′-6-FAM, 3′-H DNA oligonucleotide substrates used in gel assays); Figure S2 (unbound DNA at 70 mV applied potential); Figure S3 (primer extension gel assays supporting phi2 DNAP-DNA-dGTP ternary complex formation); Figure S4 (amplitude steps in the terminal cascade vary as a function of initial DNA substrate abasic configuration); Supporting Information Available:

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules 1-3 . Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates 4,5 . In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.We describe a nanopore device that monitors ionic current through a single protein pore inserted in a lipid bilayer (Fig. 1a). The limiting aperture of the pore is just sufficient to accommodate single-stranded DNA (ssDNA) 6,7 , and the adjacent pore vestibule can accommodate doublestranded (duplex) DNA (dsDNA) 7-9 . In the absence of DNA, the open channel current (I o ) through the α-haemolysin pore is 60 pA at 180 mV applied potential in 0.3 M KCl. DNA capture in the nanopore results in a decrease in the current (I). The DNA resides in the pore for a time (t D ) until it leaves, moving to the trans compartment (Fig. 1b). These two parameters, I and t D , together with current noise, are typically used to report results from nanopore experiments 6,10-19 .We used a nanopore instrument to probe the interaction of the Klenow fragment (KF) of Escherichia coli DNA polymerase I with its DNA substrate. This substrate is a duplex DNA formed by base-pairing of a short ssDNA primer with a longer template DNA. The KF catalyses DNA replication by the sequential addition of nucleotides to the primer strand, dictated by Watson-Crick complementarity to the template strand 20 . In contrast with earlier studies examining Exonuclease I/DNA complexes 4 and EcoRI/DNA complexes 5 , our nanopore Capture and translocation of a model DNA template (14 bp hairpin with a 36-nucleotide 5′ overhang and 2′-3′ dideoxycytidine terminus) resulted in a cluster of events with a median duration of 1 ms and an average blockade amplitude I = 20 pA (Fig. 2a). When the KF (2 μM) was subsequently added to the cis compartment under conditions where catalytic activity had been demonstrated in separate experiments (see Supplementary Information, Fig. S1), a second population of events emerged with a 3-ms median dwell time and a higher blockade current (I = 23 pA, Fig. 2b). This class of events is enzyme-concentration-dependent (see Supplementary Information, Fig. S2 and Table S1), consistent with nanopore capture of a DNA/ KF binary complex.Addition of a deoxynucleotide triphosphat...

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file order by an applied voltage. Here we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-hemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held atop the nanopore orifice at 80 mV applied potential. Sequential nucleotide additions by KF were observed upon controlled voltage reversals.