35 research outputs found
Allosteric control of the RNA polymerase by the elongation factor RfaH
Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that āfastā RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent MichaelisāMenten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 Ć
away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and Ī»Q) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex
Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins
SummaryRifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 Ć
resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation Ļ factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over ā¼19 Ć
to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal
Water vapour in the atmosphere of a transiting extrasolar planet
Water is predicted to be among, if not the most abundant molecular species
after hydrogen in the atmospheres of close-in extrasolar giant planets
(hot-Jupiters) Several attempts have been made to detect water on an exoplanet,
but have failed to find compelling evidence for it or led to claims that should
be taken with caution. Here we report an analysis of recent observations of the
hot-Jupiter HD189733b taken during the transit, where the planet passed in
front of its parent star. We find that absorption by water vapour is the most
likely cause of the wavelength-dependent variations in the effective radius of
the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger
effective radius observed at visible wavelengths may be due to either star
variability or the presence of clouds/hazes. We explain the most recent thermal
infrared observations of the planet during secondary transit behind the star,
reporting a non-detection of water on HD189733b, as being a consequence of the
nearly isothermal vertical profile of the planet.s atmosphere. Our results show
that water is detectable on extrasolar planets using the primary transit
technique and that the infrared should be a better wavelength region than the
visible, for such searches
Mechanism of molecular interactions for tRNA(Val) recognition by valyl-tRNA synthetase
The molecular interactions between valyl-tRNA synthetase (ValRS) and tRNA(Val), with the C34-A35-C36 anticodon, from Thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis. In the ValRS-bound structure of tRNA(Val), the successive A35-C36 residues (the major identity elements) of tRNA(Val) are base-stacked upon each other, and fit into a pocket on the Ī±-helix bundle domain of ValRS. Hydrogen bonds are formed between ValRS and A35-C36 of tRNA(Val) in a base-specific manner. The C-terminal coiled-coil domain of ValRS interacts electrostatically with A20 and hydrophobically with the G19ā¢C56 tertiary base pair. The loss of these interactions by the deletion of the coiled-coil domain of ValRS increased the K(M) value for tRNA(Val) 28-fold and decreased the k(cat) value 19-fold in the aminoacylation. The tRNA(Val) K(M) and k(cat) values were increased 21-fold and decreased 32-fold, respectively, by the disruption of the G18ā¢U55 and G19ā¢C56 tertiary base pairs, which associate the D- and T-loops for the formation of the L-shaped tRNA structure. Therefore, the coiled-coil domain of ValRS is likely to stabilize the L-shaped tRNA structure during the aminoacylation reaction
Atomic model of a pyrimidine dimer excision repair enzyme complexed with a dna substrate: Structural basis for damaged DNA recognition
AbstractT4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 Ć
resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60Ā° inclination at the central thymine dimer. The adenine base complementary to the 5ā² side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes
The carboxy-terminal coiled-coil of the RNA polymerase Ī²ā²-subunit is the main binding site for Gre factors
Bacterial Gre transcript cleavage factors stimulate the intrinsic endonucleolytic activity of RNA polymerase (RNAP) to rescue stalled transcription complexes. They bind to RNAP and extend their coiled-coil (CC) domains to the catalytic centre through the secondary channel. Three existing models for the GreāRNAP complex postulate congruent mechanisms of Gre-assisted catalysis, while offering conflicting views of the GreāRNAP interactions. Here, we report the GreB structure of Escherichia coli. The GreB monomers form a triangle with the tip of the amino-terminal CC of one molecule trapped within the hydrophobic cavity of the carboxy-terminal domain of a second molecule. This arrangement suggests an analogous model for recruitment to RNAP. Indeed, the Ī²ā²-subunit CC located at the rim of the secondary channel has conserved hydrophobic residues at its tip. We show that substitutions of these residues and those in the GreB C-terminal domain cavity confer defects in GreB activity and binding to RNAP, and present a plausible model for the RNAPāGreB complex