ABSTRACT
Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing â¼30% of Gcn4-binding motifs. Surprisingly, only â¼40% of the bound sites are in promoters, of which only â¼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining â¼300 Gcn4-bound sites are within coding sequences (CDSs), with â¼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.
Subject(s)
5' Flanking Region , Basic-Leucine Zipper Transcription Factors/metabolism , DNA, Fungal/metabolism , Gene Expression Regulation, Fungal , Nucleosomes/metabolism , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcriptional Activation , Basic-Leucine Zipper Transcription Factors/genetics , Binding Sites , DNA, Fungal/genetics , Histones/genetics , Histones/metabolism , Mutation , Nucleosomes/genetics , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The bacterial transcription elongation factor NusG stimulates the Rho-dependent transcription termination through a direct interaction with Rho. The mechanistic basis of NusG dependence of the Rho function is not known. Here, we describe Rho* mutants I168V, R221C/A, and P235H that do not require NusG for their termination function. These Rho* mutants have acquired new properties, which otherwise would have been imparted by NusG. A detailed analyses revealed that they have more stable interactions at the secondary RNA binding sites of Rho, which reduced the lag in initiating its ATPase as well as the translocase activities. These more stable interactions arose from the significant spatial re-orientations of the P, Q, and R structural loops of the Rho central channel. We propose that NusG imparts similar conformational changes in the central channel of Rho, yielding faster isomerization of the open to the closed hexameric states of the latter during its RNA-loading step. This acceleration stabilizes the Rho-RNA interactions at many terminators having suboptimal rut sites, thus making Rho-NusG interactions so essential in vivo Finally, identification of the NusG binding sites on the Rho hexamer led us to conclude that the former exerts its effect allosterically.
Subject(s)
Escherichia coli Proteins/metabolism , Nucleic Acid Conformation , Peptide Elongation Factors/metabolism , RNA, Bacterial/metabolism , Rho Factor/metabolism , Transcription Factors/metabolism , Transcription Termination, Genetic/physiology , Allosteric Regulation/physiology , Amino Acid Substitution , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Mutation, Missense , Peptide Elongation Factors/genetics , RNA, Bacterial/genetics , Rho Factor/genetics , Transcription Factors/geneticsABSTRACT
Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2Δ), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2Δ, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2∆, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.