Activity and nature of p21WAF1 complexes during the cell cycle

Elevated levels of the p21WAF1 (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins (“free” p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.

DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.

A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes

TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Double-level “orthogonal” dynamic combinatorial libraries on transition metal template

Dynamic combinatorial libraries are mixtures of compounds that exist in a dynamic equilibrium and can be driven to compositional self adaptation via selective binding of a specific assembly of certain components to a molecular target. We present here an extension of this initial concept to dynamic libraries that consists of two levels, the first formed by the coordination of terpyridine-based ligands to the transition metal template, and the second, by the imine formation with the aldehyde substituents on the terpyridine moieties. Dialdehyde 7 has been synthesized, converted into a variety of ligands, oxime ethers L11–L33 and acyl hydrazones L44–L77, and subsequently into corresponding cobalt complexes. A typical complex, Co(L22)22+ is shown to engage in rapid exchange with a competing ligand L11 and with another complex, Co(L22)22+ in 30% acetonitrile/water at pH 7.0 and 25°C. The exchange in the corresponding Co(III) complexes is shown to be much slower. Imine exchange in the acyl hydrazone complexes (L44–L77) is strongly controlled by pH and temperature. The two types of exchange, ligand and imine, can thus be used as independent equilibrium processes controlled by different types of external intervention, i.e., via oxidation/reduction of the metal template and/or change in the pH/temperature of the medium. The resulting double-level dynamic libraries are therefore named orthogonal, in similarity with the orthogonal protecting groups in organic synthesis. Sample libraries of this type have been synthesized and showed the complete expected set of components in electrospray ionization MS.

Topography of lacUV5 initiation complexes

Formation of a transcriptionally competent open complex is a highly regulated multistep process involving at least two intermediates. The rate of formation and stability of the intermediate complexes often determine promoter strength. However, the detailed mechanism of formation of the open complex and the high resolution structures of these intermediates are not known. In this study the structures of the open and intermediate complexes formed on the lacUV5 promoter by Escherichia coli RNA polymerase were analyzed using ‘zero length’ DNA–protein cross-linking. In both the open and the intermediate complexes the core subunits (β′ and β) interact with lacUV5 DNA in a similar way, forming DNA–protein contacts flanking the initiation site. At the same time, the recognition (σ70) subunit closely interacts with the promoter only in the open complex. In combination with our previous results, the data suggest that during promoter recognition contacts of the σ subunit with core RNA polymerase and promoter DNA are rearranged in concert. These rearrangements constitute a landmark of transition from the intermediate to the open complex, identifying the σ subunit as a key player directing formation of the open complex.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.