Protein metal-binding sites.

Metal ions have a role in a variety of important functions in proteins including protein folding, assembly, stability, conformational change, and catalysis. The presence or absence of a given metal ion is crucial to the conformation or activity of over one third of all proteins. Recent developments have been made in the understanding and design of metal-binding sites in proteins, an important and rapidly advancing area of protein engineering.     

A mechanism for passive mandibular depression

Retention of a retromandibular space has necessitated developing a system of mandibular protrusion in hominid species. It is possible that mandibular protrusion can be effected by a single muscle-the lateral pterygoid-and the motion controlled by the excentrically placed mandibular suspensory ligaments. The elasticity of the ligaments produces an integrity maintenance force between the articular condyle and eminence which is normally of minimal size. Excessive craniofacial flexion, or the retention of a juvenile configuration of the mandible, could result in increasing this integrity maintenance force and cause crepitation and clicking. Ajustment of the ligaments could reduce these pathological manifestations.     

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

Mechanistic aspects of promoter binding and chain initiation by RNA polymerase

Summary The physicochemical properties of the interactions of RNA polymerase (RPase) with promoter and nonspecific DNA sequences have been investigated. These show that nonspecific binding is principally an ionic interaction and that promoter binding is more complex, involving nonionic interactions. Nonspecific binding has been shown to be very important in the promoter search, and one-dimensional diffusion can account for the rate at which RPase finds the promoter. Significant differences have been reported in the binding process for various promoters and in the effects of regulatory proteins. Further investigation of these differences will lead to a better understanding of the selectivity and regulation of the initiation process.The pathways of the initiation process have been outlined, by recent studies and considerable progress has been made in determining the rates of interconversion of the intermediate states. A number of questions remain about the detail of initiation and the effects of various parameters on the reactions. Of particular importance is the identification of the point at which the enzyme becomes truly processive. In addition, the step which is rate limiting has not been identified in either the productive or nonproductive process. The mechanistic features of the steps after bond formation are just beginning to yield to investigation.Use of substrate analogs with RPase has led to a picture of the polymerization site according to the ability of the enzyme to incorporate analogs. Base specificity appears to be determined primarily by interaction with the template rather than the enzyme, but the ribose moiety must interact with the site quite specifically. The orientation of the phosphate residues has been determined by NMR, which has also proved to be a valuable probe of the initiation site. At this site base specificity is resident in the enzyme and expressed through the interaction of the base and intrinsic metal, as shown by studies with the Cobalt substituted enzyme. In both initiation and polymerization, the reaction has been shown to proceed by inversion of configuration. Techniques similar to those used for initiation will probably be applied to the polymerization reaction as well, which has not recently received as much attention with respect to mechanism. Functional phenomena such as pausing make the polymerization process particularly promising for producing insight into RPase reactions.     

Diversification of Hawaiian Cyrtandra (Gesneriaceae) under the influence of incomplete lineage sorting and hybridization

Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 89 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian Cyrtandra lineage, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high‐throughput sequencing, and modern phylogenomics tools to test 31 Hawaiian Cyrtandra samples (22 species, two putative hybrids, four species with two samples each, one species with four samples) and two outgroups for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species‐tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. We expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and species‐tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Multiple hybridizations were inferred, but many were on deeper branches of the island lineages suggesting a long history of hybridization. We demonstrated the utility of high‐throughput sequencing and a phylogenomic approach using 569 loci to understanding species relationships and gene flow in the presence of ILS.