Quick quantitative HSQC (QQ-HSQC) was applied to quantitative evaluation of different inter-unit linkages in an array of milled softwood and hardwood and technical lignins by using the guaiacyl C2 and syringyl C2-C6 signals as internal standards. The results were found to be highly reproducible and comparable with earlier literature reports. The advantage of QQ-HSQC NMR analysis of lignin is contemporary detection and quantification of lignin inter-unit linkages with a direct, non-destructive method requiring short acquisition times.

The lac repressor±operator system is a model system for understanding protein±DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the speci®c recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the ®rst high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of speci®c contacts between the two sites. Speci®c recognition is accomplished by a combination of elongation and twist by 48°of the right lac subunit relative to the left one, signi®cant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor±operator system is unique among other protein±DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to R-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an R-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.Eukaryotic gene transcription by RNA polymerase II requires the assembly of many proteins on the promoter region (1-3). The rate of transcription is enhanced by transcriptional activators, through recruitment of chromatin remodeling enzymes and through facilitating the assembly of the preinitiation complex (PIC) 1 (4, 5). One of the best characterized activators is the herpes simplex virion protein 16 (VP16), also known as Vmw65, ICP25, or R-TIF (6, 7). The carboxy-terminal region (residues 410-490) of this protein contains two potent transcription activation domains (TADs) (8, 9) that can target many proteins of the RNA polymerase II transcription machinery, such as TBP (10), TFIIA (11,12), TFIIB (13), the RAP74 subunit of TFIIF (14), the p62 component of TFIIH (15), the TBP-associatedfactors hTAF II 31 (16), dTAF II 40 (17), hTAF II 32 (18), the human cofactor PC4 (19,20), CBP (21,22), and p300 (23,24). The two functional regions in this acidic VP16 activation domain (VP16ad) are independently able to activate transcription in vivo via distinct pathways (8,17,22,(25)(26)(27). Both subdomains, VP16ad/n (412-453) and VP16ad/c (454-490), have been extensively studied by mutational analysis, indicating key roles for specific hydrophobic and acidic residues (26-29). The TADs of transcription factors often lack a folded structure under physiological conditions †

The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2.

The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an α-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right halfsites with different spacer lengths.