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 ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 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.

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.At the end of a significant number of operons in Escherichia coli, DNA sequences do not code for intrinsic termination signals, characterized by a hairpin and a U-stretch (1). Transcription termination factor Rho, a homo-hexameric RNA-dependent ATPase, induces termination in these operons. Rho loads onto unstructured C-rich RNA sequences (rut sites, the Rhoutilization sites) and is capable of translocating along the RNA using its RNA-dependent ATPase activity, and eventually dislodges the elongating RNA polymerase (RNAP) 3 (1, 2). By virtue of these properties of Rho, long stretches of untranslated RNA sequences are its ideal targets. Transcription on these stretches can face premature termination by Rho, which usually happens in the cases of transcription-translation uncoupling. Recent genomics study has revealed that Rho-dependent termination is widespread in vivo, which could be the main reason for its essentiality (3-6).NusG, a transcription elongation factor, being bound to the elongation complex (EC) through its N-terminal domain (NTD) helps the latter to overcome the pauses by increasing the elongation rate (5, 7, 8). Its C-terminal domain (CTD) is capable of interacting with the Rho (9). This interaction stimulates the Rho-dependent termination process in vitro, which is manifested as early termination (10, 11). More recent studies indicated that NusG is essential for recruiting Rho on a subset of terminators in vivo (4, 6). Mechanism of stimulation of Rho-dependent termination by NusG in vitro as well as the molecular basis of NusG dependence of Rho to utilize a subset of terminators in vivo is not known, knowledge of which is essential to understand the intricacies of this transcription termination process.In this study, by screening a library of ...