Transcriptional regulation involves a series of sophisticated protein-protein and protein-DNA interactions (PPI and PDI). Some transcriptional complexes, such as c-Fos/c-Jun and their binding DNA fragments, have been conserved over the past one billion years. Considering the thermodynamic principle for transcriptional complex formation, we hypothesized that the c-Fos/c-Jun complex may represent a thermodynamic summit in the evolutionary space. To test this, we invented a new method, termed One-Pot-seq, which combines cDNA display and proximity ligation to analyse PPI/PDI complexes simultaneously. We found that the wild-type c-Fos/c-Jun complex is indeed the most thermodynamically stable relative to various mutants of c-Fos/c-Jun and binding DNA fragments. Our method also provides a universal approach to detect transcriptional complexes and explore transcriptional regulation mechanisms.
DNA, Complementary , Genetic Techniques , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , DNA/metabolism , Evolution, Molecular , Mice , Multiprotein Complexes , Mutation , Polymorphism, Genetic , Protein Binding , Protein Multimerization , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Sequence Analysis, DNA , Transcription Factors/genetics
Based upon many theoretical findings on protein evolution, we proposed a ligand-selection model for the origin of proteins, in which the most ancient proteins originated from ATP selection in a pool of random peptides. To test this ligand-selection model, we constructed a random peptide library consisting of 15 types of prebiotic amino acids and then used cDNA display to perform six rounds of in vitro selection with ATP. By means of next-generation sequencing, the most prevalent sequence was defined. Biochemical and biophysical characterization of the selected peptide showed that it was stable and foldable and had ATP-hydrolysis activity as well.
Adenosine Triphosphate/chemistry , Amino Acids/chemistry , Peptide Library , Peptides/chemistry , Prebiotics , Computational Biology , DNA, Complementary/metabolism , Gene Library , High-Throughput Nucleotide Sequencing , Hydrolysis , Ligands , Protein Binding , Proteins/chemistry , RNA, Messenger/metabolism
Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting "late amino acids (C, F, M, T, W, and Y)" with "early amino acids (A, L, and V)" in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction Em,7 (fld) reached -360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity.
Amino Acids/metabolism , Megasphaera/enzymology , Mutagenesis/genetics , NADH, NADPH Oxidoreductases/metabolism , Circular Dichroism , Fluorescence , Kinetics , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Mutation/genetics , NADH, NADPH Oxidoreductases/isolation & purification , Oxidation-Reduction , Recombinant Proteins/isolation & purification
Many bacterial genomes have been sequenced and stored in public databases now, of which Reference Sequence (RefSeq) is the most widely used one. However, the annotation in RefSeq is still unsatisfactory. The present analysis is focused on the re-annotation of an important plant pathogen genome Xanthomonas oryzae pv. oryzae PXO99A (Xoo PXO99A), which is the causal agent of bacterial blight on rice. Based on the parameters of 28 nucleotide frequencies and support vector machine algorithm, 41 originally annotated hypothetical genes were recognized as noncoding sequences, which were further supported by principal component analysis and other evidence. Ten of them were tested with reverse transcription-polymerase chain reaction experiments (RT-PCR), and all of them were confirmed to be noncoding sequences. Furthermore, 197 potential new genes not annotated in RefSeq were both recognized by two ab initio gene finding programs. Most of them only have sequence similarities with part of the known genes in other species, so they are unlikely to be protein-coding genes. Twelve potential new genes have high full-length sequence similarities with function-known genes, which are very likely to be true protein-coding genes. All the 12 potential genes were tested with RT-PCR, and 11 of them (92%) were successfully amplified in cDNA template. The RT-PCR experiments confirm that our theoretical prediction has high accuracy. The improvement of Xoo PXO99A annotation is helpful for the research of lifestyle, metabolism, and pathogenicity of this important plant pathogen. The improved annotation can be obtained from http://211.69.128.148/Xoo .
Genome, Bacterial/genetics , Molecular Sequence Annotation , Oryza/microbiology , Xanthomonas/genetics , Base Sequence , DNA, Bacterial/genetics , DNA, Intergenic/genetics , Genes, Bacterial/genetics , Open Reading Frames/genetics , Polymerase Chain Reaction , Reproducibility of Results
In the title compound, C(12)H(17)NO(5)S, the orientations of the 2-ethyl-2-amino-3-hy-droxy-propano-ate group and the 4-methyl-sulfonyl moiety towards the aromatic ring are periplanar and (-)-anti-clinal, respectively. In the crystal packing, the dominant inter-action is O-Hâ¯N hydrogen bonding, which generates a chain running along [100]. N-Hâ¯O and C-Hâ¯O interactions are also observed.
In the title zwitterion, C(7)H(14)N(2)O(3), the ethyl-amino and the 5-oxo groups are positionally disordered with occupancy ratios of 0.50:0.50 and 0.70:0.30, respectively. The terminal ethyl -CH(3) group undergoes considerable thermal motion. In the crystal, mol-ecules are linked via N-Hâ¯O hydrogen bonds, forming a two-dimensional arrangement propagating in the bc plane.
