The Multicellular Effects of VDAC1 N-Terminal-Derived Peptide

The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19-26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsible for reorganization of actin and tubulin filaments, and increased cell adhesion. In addition, the peptide induced alterations in the expression of proteins associated with cell metabolism, signaling, and division, such as enhancing the expression of nuclear factor kappa B and decreasing the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha. These cellular effects may result from the peptide interfering with VDAC1 interaction with its interacting proteins, thereby blocking multiple mitochondrial/VDAC1 pathways associated with cell functions. The results of this study further support the role of VDAC1 as a mitochondrial gatekeeper protein in controlling a variety of cell functions via interaction with associated proteins

Mechanistic studies of transcription initiation by T7 RNA polymerase

Initiation of transcription is a central step in cellular regulation. In order to understand better the fundamental machanisms in this complex process, kinetic and structural studies have been carried out on a variety of modified promoter constructs using simple model RNA polymerase from bacteriophage T7. Investigation of the template strand elements in the initiation domain by introduction of non-nucleosidic spacers demonstrates that most of the apparent protein-DNA contacts in the crystal structure are not essential for initiation. In this respect, the part of the template strand from −4 to −2 serves as a “tether”, holding the templating bases near the active site. In cases where templating bases are not connected directly, the non-template strand duplexed downstream of the active site can serve a similar role. Some of the specificity in the positioning of the templating bases appears to be derived from the protein contact with the template strand base at position −1. Precise positioning of the templating bases near the active site appears to involve strong non-specific interactions of phosphodiester groups between positions −1 and +1, and +2 and +3. T7 RNA polymerase demonstrates strong preference for the initiating substrate nucleotide GTP. Experiments with the nucleotide analog 7–deaza–GTP demonstrate that this preference is due to a protein interaction with the non-Watson Crick face of the base involving the nitrogen-7 and oxygen-6 of guanine. In the presence of GTP as the sole substrate, on the template encoding GGGXX…,T7 RNA polymerase can engage in slippage transcription, making a ladder of RNA products ranging from 2 to about 14 nt in length. Termination of the ladder at 13–14 nt appears to involve a cooperative formation of structure in the nascent RNA, leading to the disruption of the transcribing complex. Slippage transcription from promoters encoding long runs of G provides evidence that the RNA:DNA heteroduplex initially can reach a length of nine nucleotides. The kinetics of synthesis of dinucleotide is characterized in a model system using numerical integration of mechanism-based rate equations. The analyses show that product rebinding cannot be ignored and can come to compete with the regular RNA production within a single enzyme turnover. Finally, the product release rate shows substrate dependence, suggesting that binding of an incoming nucleoside triphosphate can facilitate release of the bound RNA, most likely via direct competition at the active site

Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: A new kinetic model

559-56

Binding of the priming nucleotide in the initiation of transcription by T7 RNA polymerase

2819-282

Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase

21-3

The choice of start site in the initiation of transcription by T7 RNA polymerase

478A-479

The formation of structure in nascent RNA toads to termination of the G-ladder slippage product by T7 RNA polymerase

A324-A32