Charting the N-Terminal Acetylome: A Comprehensive Map of Human NatA Substrates

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

Structures and implications of TBP–nucleosome complexes

The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at gene promoters. TBP binds and bends DNA, and directs adjacent binding of the transcription factors TFIIA and TFIIB for PIC assembly. Here, we show that yeast TBP can bind to a nucleosome containing the Widom-601 sequence and that TBP–nucleosome binding is stabilized by TFIIA. We determine three cryo-electron microscopy (cryo-EM) structures of TBP–nucleosome complexes, two of them containing also TFIIA. TBP can bind to superhelical location (SHL) –6, which contains a TATA-like sequence, but also to SHL +2, which is GC-rich. Whereas binding to SHL –6 can occur in the absence of TFIIA, binding to SHL +2 is only observed in the presence of TFIIA and goes along with detachment of upstream terminal DNA from the histone octamer. TBP–nucleosome complexes are sterically incompatible with PIC assembly, explaining why a promoter nucleosome generally impairs transcription and must be moved before initiation can occur.

Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron

An evolutionarily conserved feature of introns is their ability to enhance expression of genes that harbor them. Introns have been shown to regulate gene expression at the transcription and post-transcription level. The general perception is that a promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression when in proximity to the terminator. We inserted ACT1 intron into different positions within IMD4 and INO1 genes. Transcription Run-On (TRO) analysis revealed that the transcription of both IMD4 and INO1 was maximal in constructs with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. However, activation was partially restored when the intron was placed close to the terminator. We previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through gene looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron also enhances promoter directionality and results in compact gene architecture with the promoter and terminator regions in close physical proximity. Furthermore, we show that both the promoter and terminator-proximal introns facilitate assembly or stabilization of the preinitiation complex (PIC) on the promoter. On the basis of these findings, we propose that proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron, and the terminator-proximal intron enhances transcription by affecting both the assembly of preinitiation complex and promoter directionality.

Structures of the human Mediator and Mediator-bound preinitiation complex

The 1.3-MDa transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 MDa) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation, suggesting a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the significance of TFIID in orchestrating PIC-Mediator assembly.