Mechanism of Stimulation of Ribosomal Promoters by Binding of the +1 and +2 Nucleotides

Abstract

The rate of transcription of Escherichia coli ribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions. The ؉1 initiating nucleotide and ppGpp are regulatory effectors of these promoters. The data herein show that in vitro transcription is also regulated by the ؉2 nucleotide. Both the ؉1 and ؉2 nucleotides act by driving polymerase into an altered conformation rather than by increasing the lifetime of transcription complexes. The unique design of the ribosomal promoters may stabilize a distorted state of polymerase that is relieved by the binding of the two nucleotides required for transcription initiation.Transcription of ribosomal RNA is the limiting step in ribosome production (1). The ribosomal promoters sense the overall availability of nutrients and respond by producing amounts of rRNA that can support the maximal growth possible given the nutritional environment. Nucleotides have been shown to be critical effectors of this promoter response. The signal nucleotide ppGpp is produced in inverse concentration to growth rate, and this may help keep the rate of rRNA transcription at an appropriate level (2, 3). The ribosomal promoters are also unusually sensitive to the concentration of the initiating nucleotide (4, 5). During transitions to slow growth, the nucleotide concentration can be depressed, and this prevents unnecessary transcription of ribosomal components (3).The mechanism by which nucleotides affect transcription from ribosomal promoters has received a great deal of attention. In the case of the initiating nucleotide, it was proposed that it works by stabilizing polymerase-promoter DNA complexes (4). This effect is thought to be specific to the ribosomal promoters because their complexes have short half-lives and are therefore uniquely unstable. ppGpp also reduces the complex half-life (6) and competes with the initiating nucleotide (5).There are uncertainties associated with this mechanism of nucleotide control of ribosomal transcription. In order for the short lifetime of ribosomal complexes to reduce transcription, dissociation would need to occur before the first RNA bond is formed, but this is typically quite rapid (7-10). How these unstable complexes are uniquely stabilized by the ϩ1 nucleotide is not known. In addition, recent experiments in other transcription systems have shown that the ϩ2 nucleotide can also have stimulatory effects. This occurs during initiation by the viral T7 RNA polymerase (11) and during elongation by the Escherichia coli RNA polymerase (12). Nucleotides that do not match the ϩ1 position on the template can stimulate the isomerization of E. coli RNA polymerase in complexes with fork-junction DNA.1 The structure of such complexes is known (13) and gives no obvious clue as to how this stabilization by nucleotides could occur.For these reasons we have re-evaluated the role of nucleotides as effectors of ribosomal transcription. The new data show that both the ϩ1 and ϩ2 nucleotides can act as effectors, unifying the properties of...

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

“…We found that the requirement for high concentrations of initiating NTPs was specific for an initiation step prior to or including first phosphodiester bond formation. By altering the initiating (ϩ1 and ϩ2) nucleotides of several promoters we observed that, as for bacterial rRNA promoters (20,21), both the ϩ1 and ϩ2 nucleotides were very important for promoter utilization. In fact, like T7 RNAP (22) we found that the NTP used for the ϩ2 position of the transcript was needed at higher concentration than the ϩ1 NTP.…”

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

“…These views are not mutually exclusive and in fact, the properties of DksA support both, as it not only destabilizes the ribosomal complexes and but also raises the K m for initiating nucleotides (Paul et al ., 2004b). This might be expected if the high K m and short lifetime were a common outcome of the effect of unusual ribosomal promoter elements on RNA polymerase structure (Lew and Gralla, 2004a). …”

“…It also binds the NTPs with an usually high K m . 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).…”

“…Three features of the core promoter are unusual: a short spacer that separates near consensus -10 and -35 elements; a CG-rich 'discriminator' sequence just upstream from + 1 (Pemberton et al ., 2000) and a long distance between the -10 region element and the + 1 start site. The specificity for nucleotide regulation might lie in the conjunction of these unusual promoter features (Lew and Gralla, 2004a).…”

“…Another property of the ribosomal promoters also contributes to regulation by nucleotide; they have an unusually high K m for initiation by the + 1 (see Schneider et al ., 2002) and + 2 NTPs (Lew and Gralla, 2004a) that form the 5 ¢ end of the ribosomal transcript. This property allows selective inhibition of rRNA transcription when NTP concentrations fall.…”