ABSTRACT
Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5'-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5' end. Thus, the RNA 5' end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5'-exonucleases during early transcription.
Subject(s)
RNA Caps , RNA Precursors/genetics , RNA, Fungal/genetics , Transcription, Genetic , Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Cryoelectron Microscopy , Mass Spectrometry , Models, Genetic , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nucleic Acid Conformation , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Phosphorylation , Protein Binding , Protein Structure, Quaternary , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA Splicing , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
For transcription elongation, all cellular RNA polymerases form a stable elongation complex (EC) with the DNA template and the RNA transcript. Since the millennium, a wealth of structural information and complementary functional studies provided a detailed three-dimensional picture of the EC and many of its functional states. Here we summarize these studies that elucidated EC structure and maintenance, nucleotide selection and addition, translocation, elongation inhibition, pausing and proofreading, backtracking, arrest and reactivation, processivity, DNA lesion-induced stalling, lesion bypass, and transcriptional mutagenesis. In the future, additional structural and functional studies of elongation factors that control the EC and their possible allosteric modes of action should result in a more complete understanding of the dynamic molecular mechanisms underlying transcription elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Subject(s)
RNA Polymerase II/chemistry , Transcription Elongation, Genetic/physiology , Transcriptional Elongation Factors/chemistry , Animals , DNA Damage/genetics , DNA Damage/physiology , Humans , Models, Biological , Models, Molecular , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/physiology , Structure-Activity Relationship , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/physiologyABSTRACT
Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , DNA-Directed RNA Polymerases/chemistry , Pyrococcus furiosus/chemistry , Pyrococcus furiosus/enzymology , Transcriptional Elongation Factors/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Repressor Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Sequence Homology, Amino AcidABSTRACT
Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.
