Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases.

Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single ß-α-ß unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined ß-α-ß units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These "fused" 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or ß-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and ßArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and ß) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.

Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications.

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple ß-α-ß structural scaffold. Most members are constructed from a single-core ß-α-ß motif or two consecutively fused ß-α-ß motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry.

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and its phosphorylation state are critically important in regulating transcription in vivo. Early methods of identifying phosphorylation on the CTD heptad were plagued by issues of low specificity and ambiguous signals. However, advancements in the field of mass spectrometry (MS) have presented the opportunity to gain new insights into well-studied processes as well as explore new frontiers in transcription. By using MS, residues which are modified within the CTD heptad and across repeats are now able to be pinpointed. Likewise, identification of kinase and phosphatase specificity towards residues of the CTD has reached a new level of accuracy. Now, MS is being used to investigate the crosstalk between modified residues of the CTD and may be a critical technique for understanding how phosphorylation plays a role in the new LLPS model of transcription. Herein, we discuss the development of various MS techniques and evaluate their capabilities. By highlighting the pros and cons of each technique, we aim to provide future investigators with a comprehensive overview of how MS can be used to investigate the complexities of RNAP-II mediated transcription.