Effect of salt and RNA structure on annealing and strand displacement by Hfq

The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq''s RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA. The rate of strand exchange between the oligonucleotides increased 80-fold. Therefore, Hfq is very active in both helix formation and exchange. However, high concentrations of Hfq destabilize the duplex by preferentially binding the single-stranded RNA. RNA binding and annealing were completely inhibited by 0.5 M salt. The target site in DsrA sRNA was 1000-fold less accessible to the molecular beacon than an unstructured oligonucleotide, and Hfq accelerated annealing with DsrA only 2-fold. These and other results are consistent with recycling of Hfq during the annealing reaction, and suggest that the net reaction depends on the relative interaction of Hfq with the products and substrates.     

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).     

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼10⁸ M⁻¹s⁻¹) observed with Cy3-Hfq was followed by a slow transition (0.5 s⁻¹) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.     

Qβ噬菌体RNA复制酶基因的克隆及在大肠杆菌中的表达

构建了带有Qβ噬菌体RNA复制酶cDNA基因的重组表达质粒pKK-Rep,采用电泳分析的方法考察了不同浓度的IPTG、不同的葡萄糖浓度对pKK-Rep在E.coli JM109中表达RNA复制酶蛋白的影响。结果表明,0.01mmol/L IPTG可以有效诱导pKK-Rep表达RNA复制酶蛋白,但表达的RNA复制酶蛋白大多数为不溶性蛋白。在培养基中添加0.02%的葡萄糖对该RNA复制酶蛋白的组成型表达无明显的影响,但当葡萄糖浓度增加至0.04%-1.0%时,这种组成型表达量显著降低。采用MDV－poly( )RNA作为模板来体外检测可溶性表达蛋白的活性,结果表明其具有体外特异扩增RNA模板的活性。     

Thermal transitions in E. coli +RNA fMet and two of its molecular fragments

Melting curves of tRNA^fMet and two fragments derived from this molecule by limited ribonuclease T1 digestion (i.e., the anticodon arm and loop [K fragment] and the larger fragment representing three-fourths of the tRNA chain from the 3′ terminus including two potential limbs of the cloverleaf structure [L fragment]) are presented. The profiles observed are consistent with the presence of base paired structures in all those molecules. At low salt concentration (0.02M Na⁺) the stabilities of these molecules measured by the apparent midpoints of the denaturation profiles are in the order K > L > tRNA. The relative stabilities approach each other at 0.2M Na⁺ (the tRNA profile being biphasic), while at high salt (2M) the L fragment seems to be more stable than either K or t-RNA ^fMet. Estimation of the enthalpy of denaturing the K structure in 0.02M Na⁺ gives a value of 40 ± 3 kcal/mole corresponding to an enthalpy per effective G.C. base pair disrupted of 10 ± 1 kcal/mole.     

Stability and maturation of E. coli ribosomal RNA.

The kinetics of rRNA maturation were investigated in a rifampicin permeable strain of E. coli during exponential growth in glucose minimal medium. The method used involves isotopic labelling of rRNA, and separation of precursor and mature forms by gel electrophoresis. The maturation of both 16s and 23s rRNA was found to follow first order kinetics. The mean life time of the precursors was found to be about 1.5 min. In glucose minimal medium all pulse label in precursors was recovered in mature rRNA, i.e. nascent rRNA is stable.     

The kinetics of E. coli RNA polymerase.

Using an assay specific for chain elongation of E. coli RNA polymerase the kinetics of this propagation reaction have been studied. The kinetic behaviour is consistent woth the mathematical model formulated for this multisubstrate enzyme. The effect of increasing salt concentration on the kinetics of the reaction indicated that DNA unwinding is probably a necessary step in the propagation step, although this may not be the rate limiting step under all conditions.     

Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

Escherichia coli UvrD is an SF1A (superfamily 1 type A) helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded DNA (ssDNA) translocase but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500?μM), we proposed a nonuniform stepping mechanism in which a UvrD monomer translocates with biased (3' to 5') directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step size of 4-5?nt/step, suggesting that a pause occurs every 4-5?nt translocated. To further test this mechanism, we examined UvrD translocation over a range of lower ATP concentrations (10-500?μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ～1 ATP/DNA base translocated even at the lowest ATP concentration examined (10?μM), indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4-5?nt/step down to 50?μM ATP but increases to ～7?nt/step at 10?μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis.     

A new gene in E. coli RNA synthesis

A novel spontaneous temperature sensitive mutant of Escherichia coli, which stops synthesizing stable RNA and some proteins immediately upon temperature shift from 30 degrees C to 42 degrees C, is described. Stable RNA species are not preferentially degraded in the mutant at the nonpermissive temperature. The guanine polyphosphate compounds, ppGpp (MS1) and pppGpp (MS2), are not produced at 42 degrees C. The mutant strain does not grow at 42 degrees C in either broth or defined minimal medium supplemented with any of a variety of carbon sources. The temperature sensitive mutation in this strain maps between dap A, E and pts I and defines a new locus affecting RNA synthesis in E. coli.