Regulation through the Secondary Channel—Structural Framework for ppGpp-DksA Synergism during Transcription

Perederina, Anna; Svetlov, Vladimir; Vassylyeva, Marina N.; Tahirov, T.H.; Yokoyama, Shigeyuki; Artsimovitch, Irina; Vassylyev, Dmitry G.

doi:10.1016/j.cell.2004.06.030

Cited by 322 publications

(468 citation statements)

References 42 publications

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“…13; I. Toulokhonov, J. Mukhopadhyay, R. H. Ebright, and R.L.G., unpublished data). A model has been proposed in which DksA coordinates the Mg 2ϩ ion bound to the diphosphate of ppGpp facing the outlet to the secondary channel (13).…”

Section: Effects Of Dksa On Positive Regulation Of Amino Acid Promotementioning

confidence: 84%

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DksA potentiates direct activation of amino acid promoters by ppGpp

Paul

Berkmen

Gourse

2005

Proc. Natl. Acad. Sci. U.S.A.

298

414

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Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp͞ DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.RNA polymerase ͉ rRNA transcription ͉ transcription initiation ͉ amino acid biosynthesis ͉ stringent response A mino acid starvation in Escherichia coli results in global changes in gene expression called the ''stringent response'' (reviewed in ref. 1). This regulatory response is initiated by entry of uncharged tRNAs into the A site of the ribosome and activation of the ribosome-associated RelA protein to synthesize the ''alarmone'' ppGpp (used here to refer to both guanosine 5Ј-diphosphate 3Ј-diphosphate and its pentaphosphate precursor). During starvation for amino acids, expression of stable RNA (rRNA and tRNA) is inhibited, whereas expression of enzymes for amino acid biosynthesis and transport is induced. These negative and positive responses are both relA dependent (e.g., refs. 2-6).ppGpp directly and specifically inhibits the initiation of transcription from stable RNA promoters in vivo and in vitro (7)(8)(9)(10)(11)(12). Although the details of the mechanism by which ppGpp exerts its effects on transcription initiation are still ill-defined, it has been shown that ppGpp decreases the lifetimes of competitorresistant complexes between RNA polymerase (RNAP) and all promoters that have been examined (12). The complex formed by RNAP at rRNA promoters is intrinsically short-lived, and ppGpp further decreases the lifetime of this complex. Thus, we have proposed that ppGpp shifts the equilibriu...

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Section: Effects Of Dksa On Positive Regulation Of Amino Acid Promotementioning

confidence: 84%

“…Rather, DksA binds in the secondary (NTP) channel of RNAP (13), thereby decreasing the lifetime of competitor-resistant complexes and the concentration of ppGpp required for inhibiting rRNA transcription. It also greatly enhances inhibition of transcription from rRNA promoters by ppGpp in vitro (11).…”

mentioning

confidence: 99%

DksA potentiates direct activation of amino acid promoters by ppGpp

Paul

Berkmen

Gourse

2005

Proc. Natl. Acad. Sci. U.S.A.

298

414

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“…19 DksA exhibits an overall similarity in structure to the transcription elongation factors GreA and GreB, despite a lack of sequence homology. The Gre factors bind in the secondary channel of RNAP, a pore though which NTP substrates gain access to the active site of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…Like the Gre factors, DksA prevents the formation of arrested complexes, although it cannot activate the intrinsic cleavage activity of RNAP or rescue arrested elongation complexes. 19 To provide further insight into the mechanism of DksA function, we compared the effects of DksA and the Gre factors on transcription initiation in vitro and in vivo. If one or both Gre factors could perform the same functions as DksA, this would suggest that a shared property of the factors might explain the effects of DksA.…”

Section: Introductionmentioning

confidence: 99%

Effects of DksA, GreA, and GreB on Transcription Initiation: Insights into the Mechanisms of Factors that Bind in the Secondary Channel of RNA Polymerase

Rutherford

Lemke

Vrentas

et al. 2007

Journal of Molecular Biology

110

183

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SummaryEscherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfill some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentrations of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When overexpressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA only modestly affected rRNA promoters in vitro, and even when overexpressed GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the ω subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

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“…As suggested earlier, DksA, whose cellular concentration does not change significantly under different growth conditions, seems primarily to augment the inhibitory effect of ppGpp and also compensates for the loss of the v subunit of RNA polymerase in the response of rRNA promoters to ppGpp (Rutherford et al, 2007). Additional detailed studies suggest that DksA, which binds to the secondary channel of RNA polymerase, interferes allosterically with the transcription initiation site, affecting the transition from Combined effects of ppGpp, DksA and the iNTPs during stringent control Both ppGpp and DksA exert their effect by direct binding to the secondary channel of RNA polymerase (Artsimovitch et al, 2004;Perederina et al, 2004). Both molecules are directly located near the active centre, where nucleotides are incorporated, although some of the conclusions with respect to ppGpp and E. coli RNA polymerase have been challenged by a controversial study (Vrentas et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides

et al. 2011

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Transcription of rRNAs in Escherichia coli is directed from seven redundant rRNA operons, which are mainly regulated by their P1 promoters. Here we demonstrate by in vivo measurements that the amounts of individual rRNAs transcribed from the different operons under normal growth vary noticeably although the structures of all the P1 promoters are very similar. Moreover, we show that starvation for amino acids does not affect the seven P1 promoters in the same way. Notably, reduction of transcription from rrnD P1 was significantly lower compared to the other P1 promoters. The presence of DksA was shown to be crucial for the ppGpp-dependent downregulation of all P1 promoters. Because rrnD P1 is the only rrn promoter starting with GTP instead of ATP, we performed studies with a mutant rrnD promoter, where the initiating G+1 is replaced by A+1. These analyses demonstrated that the ppGpp sensitivity of rrn P1 promoters depends on the nature and concentration of initiating nucleoside triphosphates (iNTPs). Our results support the notion that the seven rRNA operons are differentially regulated and underline the importance of a concerted activity between ppGpp, DksA and an adequate concentration of the respective iNTP. INTRODUCTIONThe rapid adaptation of cell growth in response to changing environmental conditions is central for the fitness and survival of bacteria. Bacterial growth rate is largely determined by the number of ribosomes. Hence, regulation of the translational components plays a central role in adaptation. The biogenesis of ribosomes, representing one of the cell's most energy-intensive activities, is governed by a complex network of regulation (Schneider et al., 2003;Wagner, 2009). The key molecules in this network are rRNAs. In contrast, the synthesis of ribosomal proteins, despite having similar regulatory mechanisms to those for rRNA, is largely linked to rRNA transcription by a translational feedback mechanism, which couples ribosomal protein translation to the amount of available rRNAs (Keener & Nomura, 1996;Lemke et al., 2009;Nomura et al., 1984). Therefore, the precise adjustment of rRNA synthesis rate to the nutritional quality and supply from the environment ensures that an appropriate number of ribosomes will be produced and energy resources are not wasted (Condon et al., 1995b;Wagner, 1994Wagner, , 2009).In Escherichia coli, rRNA genes (16S, 23S and 5S rRNA), together with at least one tRNA gene, are organized in a redundant fashion in seven different transcriptional units (the rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH operons). Although the seven ribosomal operons exhibit a high structural similarity, there are several striking sequence micro-heterogeneities, which could potentially contribute to functionally different ribosome populations (Wagner, 2009). Evidence for such specialized ribosomes in various organisms has been presented (Gunderson et al., 1987;Kim et al., 2007; Ló pez-Ló pez et al., 2007). If the different rRNA operons encode functionally distinct molecules one would expect tha...

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Regulation through the Secondary Channel—Structural Framework for ppGpp-DksA Synergism during Transcription

Cited by 322 publications

References 42 publications

DksA potentiates direct activation of amino acid promoters by ppGpp

DksA potentiates direct activation of amino acid promoters by ppGpp

Effects of DksA, GreA, and GreB on Transcription Initiation: Insights into the Mechanisms of Factors that Bind in the Secondary Channel of RNA Polymerase

Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides

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