Classification of doubly wound nucleotide binding topologies using automated loop searches

A classification is presented of doubly wound α/β nucleotide binding topologies, whose binding sites are located in the cleft formed by a topological switch point. In particular, the switch point loop nearest the N-terminus is used to identify specific structural classes of binding protein. This yields seven structurally distinct loop conformations, which are subsequently used as motifs for scanning the Protein Data Bank. The searches, which are effective at identifying functional relationships within a large database of structures, reveal a remarkable and previously unnoticed similarity between the coenzyme binding sites of flavodoxin and tryptophan synthetase, even though there is no sequence or topological similarity between them.     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

Porcine growth hormone gene expression from viral promoters in transgenic swine

Abstract

The production of porcine growth hormone (pGH) from novel expression vectors containing the promoter/enhancer elements of the Moloney murine leukemia virus (MLV) LTR or the human cytomegalovirus (CMV) immediate early gene was examined in transgenic swine. Both fusion genes resulted in elevated levels of serum pGH, elevation of insulin‐like growth factor 1 (IGF‐1), and a pronounced decrease in carcass fat deposition. The two viral promoter/enhancer elements were constitutively active in the transgenic swine throughout the life of the animals. In individual swine, the CMV‐pGH transgene was expressed predominantly in the pancreas while the MLV‐pGH transgene was expressed in a wide variety of tissues. These swine were infertile, had insulin resistance, and demonstrated an accelerated form of osteochondritis dissicans. Our results show that excess pGH produces a phenotype identical to that seen in swine expressing heterologous growth hormones, and provides a baseline for assessing the overall efficiency of producing transgenic swine. Furthermore, our data suggests that unregulated pGH production, even at 15 times normal levels and independent of the tissue source, has adverse effects that outweigh the desired reduction in carcass fat deposition in transgenic swine.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs

Type I collagen is composed of two α1(I) polypeptides and one α2(I) polypeptide and is the most abundant protein in the human body. Expression of type I collagen is primarily controlled at the level of mRNA stability and translation. Coordinated translation of α(I) and α2(I) mRNAs is necessary for efficient folding of the corresponding peptides into the collagen heterotrimer. In the 5' untranslated region (5' UTR), collagen mRNAs have a unique 5' stem-loop structure (5' SL). La ribonucleoprotein domain family member 6 (LARP6) is the protein that binds 5' SL with high affinity and specificity and coordinates their translation. Here we show that RNA helicase A (RHA) is tethered to the 5' SL of collagen mRNAs by interaction with the C-terminal domain of LARP6. In vivo, collagen mRNAs immunoprecipitate with RHA in an LARP6-dependent manner. Knockdown of RHA prevents formation of polysomes on collagen mRNAs and dramatically reduces synthesis of collagen protein, without affecting the level of the mRNAs. A reporter mRNA with collagen 5' SL is translated three times more efficiently in the presence of RHA than the same reporter without the 5' SL, indicating that the 5' SL is the cis-acting element conferring the regulation. During activation of quiescent cells into collagen-producing cells, expression of RHA is highly up-regulated. We postulate that RHA is recruited to the 5' UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of the most abundant protein in the human body.     

Protein structure preference, tRNA copy number, and mRNA stem/loop content

From statistical analyses of protein sequences for humans and Escherichia coli we found that the messenger RNA segment of m-codons (for m=2 to 6) with average high tRNA copy number (TCN) (larger than approximately 10.5 for humans or approximately 1.95 for E. coli) preferably code for the alpha helix and that with low TCN (smaller than approximately 7.5 for humans or approximately 1.7 for E. coli) preferably code for coil. Between them there is an intermediate region without correlation to structure preference. For the beta strand the preference/ avoidance tendency is not obvious. All strong preference-modes of TCN for protein secondary structures have been deduced. The mutual interaction between two factors--protein secondary structural type and codon TCN--is tested by F distribution. A phenomenological model on the relation between structure preference and translational efficiency or accuracy is proposed. It is pointed out that the structure preference of codons is related to the distribution of mRNA stem/loop content in three TCN regions.     

Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease‐free planting materials is essential for preventing disease development in the field. In this study, a species‐specific loop‐mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species‐specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100‐fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut‐disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut‐free seedcanes. This is the first report of LAMP‐based assay for the detection of S. scitamineum in sugarcane.     

Role of the 207-218 peptide region of Moloney murine leukemia virus integrase in enzyme catalysis

X-ray diffraction data on a few retroviral integrases show a flexible loop near the active site. By sequence alignment, the peptide region 207-218 of Mo-MLV IN appears to correspond to this flexible loop. In this study, residues H208, Y211, R212, Q214, S215 and S216 of Mo-MLV IN were mutated to determine their role on enzyme activity. We found that Y211A, R212A, R212K and Q214A decreased integration activity, while disintegration and 3′-processing were not significantly affected. By contrast H208A was completely inactive in all the assays. The core domain of Mo-MLV integrase was modeled and the flexibility of the region 207-216 was analyzed. Substitutions with low integration activity showed a lower flexibility than wild type integrase. We propose that the peptide region 207-216 is a flexible loop and that H208, Y211, R212 and Q214 of this loop are involved in the correct assembly of the DNA-integrase complex during integration.