Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex

Abstract. Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and exportbound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran–GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion o

N-terminal acetylation and protonation of individual hemoglobin subunits: Position-dependent effects on tetramer strength and cooperativity

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the α- or the β-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the α-subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer–dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N-terminals of the β-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer–dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the α-subunit, the slope of the plot of the tetramer–dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the α-subunits is less extensive than that of the β-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer–dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic ζ-subunits leads to an average 20-fold weakening of tetramer–dimer interfaces compared to corresponding hemoglobins containing adult α-subunits. The dimer–monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain ζ-subunits and exchange with added β-subunits in the form of β4 (HbH) significantly faster than do those with α-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (ζ2β2) < Hb Portland-1 (ζ2γ2) ≅ Hb Gower-1 (ζ2ɛ2) < Hb Gower-2 (α2ɛ2) < HbF1 < HbF (α2γ2) < HbA2 (α2δ2), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer–monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic → fetal → adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks