Nucleotide-Dependent Isomerization of Escherichia coli RNA Polymerase

Lew, Chih M.; Gralla, Jay D.

doi:10.1021/bi0492814

Cited by 9 publications

(6 citation statements)

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“…Both the ϩ1 and ϩ2 nucleotides of bacterial rRNA promoters are also required at high concentrations to stabilize the initiation complex (35) and possibly drive the isomerization of the polymerase into an initiation-competent conformation (21,36). These initiation-specific NTP requirements are responsible for changes in ribosomal RNA transcriptional patterns that are sensitive to fluctuations in intracellular ATP and GTP concentrations (35).…”

Section: Discussionmentioning

confidence: 99%

Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides

Amiott¹,

Jaehning²

2006

Journal of Biological Chemistry

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Despite a simple consensus sequence, there is considerable variation of promoter strengths, transcription rates, and the kinetics of initiating nucleotide incorporation among the promoters found in the Saccharomyces cerevisiae mitochondrial genome. We asked how changes in the initiating (؉1 and ؉2) nucleotides, conformation of the promoter DNA template, and mutation of the mitochondrial RNA polymerase (mtRNAP) affect the kinetics of nucleotide (NTP) utilization. Using a highly purified in vitro mitochondrial transcription system, we found that 1) the mtRNAP requires the highest concentrations of the ؉1 and ؉2 initiating NTPs, intermediate concentrations of NTPs at positions 5 to 11, and low concentrations of elongating NTPs; 2) the mtRNAP requires a higher concentration of the ؉2 NTP than the ؉1 NTP for initiation; 3) the kinetics of ؉2 NTP utilization are altered by a point mutation in the mtRNAP subunit Mtf1; and 4) a supercoiled or pre-melted promoter DNA template restores normal ؉2 NTP utilization by the Mtf1 mutant. Based on comparisons to the structural and biochemical properties of the bacterial RNAP and the closely related T7 RNAP, we propose that initiating nucleotides, particularly the ؉2 NTP, are required at high concentrations to drive mitochondrial promoter opening or to stabilize a productive open complex.The core subunit of the budding yeast mitochondrial RNA polymerase (mtRNAP), 3 encoded by the nuclear gene RPO41, is a 145-kDa protein that shares significant amino acid similarity with the T7/T3 bacteriophage family of single subunit RNAPs (1, 2). However, T7 RNAP is a single subunit enzyme capable of specific and regulated activity in the absence of any transcription factors (3), but mtRNAPs from all eukaryotes examined to date require one or more auxiliary factors for selective transcription (reviewed in Ref. 4). The accessory factor for the yeast mtRNAP, Mtf1, is a 43-kDa protein that interacts directly with Rpo41, creating a functional "holoenzyme" with promoter-specific activity (5). Following the synthesis of a short RNA chain (ϳ12 nucleotides), Mtf1 is released, and Rpo41 continues elongation of the transcript (6).Recently it has been demonstrated that Rpo41 alone can initiate transcription in a promoter-specific manner in vitro in the absence of Mtf1 (7). However, this Mtf1-independent activity is only possible on supercoiled or pre-melted "bubble" promoter templates in which the promoter opening stages of transcription initiation have been facilitated or bypassed. Mtf1 alters the ratio of full-length to abortive transcripts on nonlinear templates (7) demonstrating that Mtf1 influences both promoter opening and clearance by the mtRNAP. Despite these findings, the mechanistic function of Mtf1 and its homologs in promoter selective transcription is still not well understood.The yeast mtRNAP mediates gene expression in the mitochondrial compartment by binding and initiating transcription at a collection of short, nonanucleotide (consensus ATATA-AGTA) promoters encompassing the transcriptiona...

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Section: Discussionmentioning

confidence: 99%

Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides

Amiott¹,

Jaehning²

2006

Journal of Biological Chemistry

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“…It has been proposed that both properties are a common outcome of the unusual promoter elements imposing a distorted conformation on the bound polymerase (Lew and Gralla, 2004a). Ribosomal promoters might require NTP binding to complete the isomerization of polymerase to its functional form (Lew and Gralla, 2004b). Whatever the details, these two properties likely make ribosomal transcription preferentially sensitive to a decline in NTP concentration.…”

Section: Molecular Mechanism Of Regulationmentioning

confidence: 99%

Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase‐binding protein

Gralla

2005

Molecular Microbiology

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SummaryRibosomal RNA transcription can limit the rate of Escherichia coli growth and is subject to complex regulation. Somehow, the cell is able to sense the general nutritional environment and adjust rRNA transcription so that an appropriate number of ribosomes is produced. This review discusses the current state of affairs, including recent information about the involvement of two nucleotide regulators, two architectural protein regulators, one new co-regulator and stalled ribosomes.

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“…It is known that binary open (RP o ) complexes of the stringent-sensitive rRNA P1 promoters are intrinsically unstable, and only after binding of the first substrate NTP are stable kinetic intermediates (RP i ) formed (Barker et al, 2001;Gourse, 1988;Langert et al, 1991). Consistently, the K m for the incorporation of the first NTPs is unusually high for the stringent-sensitive rrn promoters, explaining the requirement for high concentrations of the iNTPs (ATP and GTP) for efficient transcription initiation (Lew & Gralla, 2004;Schneider et al, 2002). The presence of ppGpp further reduces the lifetime of the intrinsically short-lived open promoter complexes (Barker et al, 2001) and it is proposed that DksA binding to RNA polymerase affects the active site, interfering with NTP addition (Rutherford et al, 2009).…”

Section: Discussionmentioning

confidence: 93%

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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Nucleotide-Dependent Isomerization of Escherichia coli RNA Polymerase

Cited by 9 publications

References 56 publications

Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides

Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides

Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase‐binding protein

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

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