The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.

The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. coli. These RNA sequences each carry a rho "loading site" at a position near the 5'-end, and a rho-dependent terminator sequence at the 3'-end to which complementary approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3'-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.

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The strychnine-sensitive glycine receptor, a member of a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system, forms an anion-specific transmembrane channel in response to glycine binding. A rapid-reaction technique, a cell-flow method with a 10-ms time resolution, was adapted for measurements with cultured embryonic mouse spinal cord cells containing glycine receptors. Whole-cell current responses resulting from the opening of glycine receptor channels were measured at pH 7.4, 22-24 degrees C, and transmembrane voltages of -40 and -75 mV. Two different receptor forms, A alpha and A beta, were detected. At saturating glycine concentrations, an average of 70% of the whole-cell current amplitude was associated with form A alpha and 30% with A beta. The constants pertaining to the minimum mechanisms that account for the concentration of the two open-channel receptor forms over a 100-fold range of glycine concentration were determined by cell-flow measurements of the current amplitudes and of the falling (desensitizing) rate of the current. The dissociation constant of the site controlling channel opening was 220 microM on the basis of three binding sites for A alpha and 380 microM on the basis of two binding sites for A beta. The channel-opening equilibrium constant, phi-I, was 170 for A alpha and 110 for A beta. The rate coefficients for desensitization, alpha and beta, associated with these two forms have maximum values of 0.7 and 0.1 s-1, respectively. The rates at which the receptors recovered from desensitization were also measured, using a double-flow mixing device, and were found to be 0.06 s-1 for A alpha and 0.02 s-1 for A beta. In the presence of 100 microM glycine, the apparent dissociation constant for the inhibitor picrotoxinin from receptor form A alpha was 80 microM, and that from A beta was 460 microM. This suggests that A beta contains beta-subunits (58 kD), because this subunit confers picrotoxinin insensitivity to glycine receptors (Pribilla, I., et al. (1992) EMBO J. 11, 4305). In the case of one receptor form (A alpha), the chemical mechanism and its constants led to two measurements that could be assessed by an independent method, the single-channel current-recording technique: (i) the fraction of receptor channels open at a given glycine concentration (ALn)o and (ii) the rate coefficient for desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)