The sinus node is a collection of highly specialised cells constituting the heart’s pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca 2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.

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Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

Protein synthesis in ‘minicells’ showed that the DNA immediately preceding the pyrE gene of Escherichia coli directs the formation of considerable amounts of a polypeptide (mol. wt. approximately 30 000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream to the structure start of pyrE, just prior to the GC‐rich symmetry region of a sequence with features characteristic of a rho‐independent transcription terminator. Deletion of the start of this 238 codons long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that the two genes (ORF and pyrE) constitute an operon. S1‐nuclease digestion of RNA‐DNA hybrids revealed that both genes are transcribed from two promoters (P1 and P2) located in front of the ORF start. Furthermore, when the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was found to be constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. From these observations we conclude that pyrE expression is controlled by a UTP modulated intercistronic attentuation.

We have used the galK gene, minus its promoter, to quantitate transcription of the orfE--pyrE operon of Escherichia coli in front of and after the intercistronic attenuator. Expression of the hybrid genes was studied in a bacterium with mutations that permit changes in the UTP and GTP pools during exponential growth. It was found that the greater part of pyrE gene regulation by the nucleotides takes place at the intercistronic attenuator and that promoter control contributes only little, ca. twofold. When pools of both UTP and GTP were high only 5%-6% of the mRNA chains were continued into the pyrE gene. However, when the UTP pool was reduced (from 1.3 to 0.2 mumol/g dry weight) nearly 100% of transcription passed the attenuator. Likewise, a reduction in the GTP pool (from 3.2 to 0.8 mumol/g dry weight) resulted in 25%-30% escape of attenuation. Regulation by attenuation disappeared when a premature stop-codon was introduced near the end of orfE such that translational coupling to transcription was prevented in the attenuator area. Therefore, we attribute the modulation of attenuation to nucleotide-induced variations in the kinetics of mRNA chain elongation. In support for this it was found that an RNA polymerase mutant with reduced RNA chain growth rate transcribed past the pyrE attenuator at a high frequency in the presence of a high UTP pool, but only when coupling of translation to transcription was allowed at the end of orfE.

Orotate phosphoribosyltransferase (EC 2.4.2.10) was purified to electrophoretic homogeneity from a strain of Escherichia coli containing t h e p y r E gene cloned o n a multicopy plasmid. The relative molecular masses (M,) of the native enzyme and its subunit were estimated by means of gel filtration and electrophoresis in the presence of dodecyl sulfate. The amino acid sequences at the N and C termini, as well as the amino acid composition, were determined.The nucleotide sequence of the structuralpyrE gene, including 394 nucleotide residues preceding the beginning of the coding frame, was also established. From the results the following conclusions may be drawn.Orotate phosphoribosyltransferase is a dimeric protein with subunits of M, 23 326 consisting of 21 1 amino acid residues. The pyrE gene is transcribed in a counter-clockwise direction from the E. coli chromosome as an mRNA with a considerable leader segment in front of the protein-coding region. This leader contains a structure with features characteristic for a (translated ?) rho-independent transcriptional terminator, which is preceded by a cluster of uridylate residues. This indicates that the frequency ofpyrE transcription is regulated by an R N A polymerase (UTP) modulated attenuation Orotate phosphoribosyltransferase (EC 2.4.2.10) catalyses one of the reactions in the de nova synthesis of UMP (Fig. 1). The pyr genes are scattered on the chromosome of enteric bacteria, and the structural gene for orotate phosphoribosyltransferase, pyrE, is located at 81 min o n the Eschrrichia coli linkage map, between dut and spoT [I].Both in E. coli and in Sulmonella typhimuriurn the expression of p y B , pyrE, and p y r F is repressed by a uridine nucleotide, which is U T P o r UDP [2-41, while it is elevated by partial guanine starvation [ 5 , 61. True regulatory mutants defective in the control of pyr gene activity have not been identified. However, it has been found that some R N A polymerase mutants (rpoBC) display high, constitutive pyrB and pyrE expression, while at the same time they contain high intracellular concentrations of uridine nucleotides 171. All other identified mutants with enhanced expression of the pyr genes have been shown to harbor defects in nucleotide interconversion pathways (pyrH, guaB) [S, 81. These observations indicate that R N A polymerase is involved in sensing the regulatory signals in pyr gene regulation. To characterize the biochemistry behind the control of pyrimidine biosynthesis further we have initiated a study of the individual pyr genes. This paper describes the nucleotide sequence of the E. coli p y r E gene and of its regulatory region.This gene had previously been cloned onto various multicopy plasmids [9 -111, and from a strain containing one of these [I03 we have purified the gene product, orotate phosphoribosyltransferase, and characterized its structure in some detail. The same plasmid served as a source of D N A restriction fragments for D N A sequencing. The results indicate that t h e p y r E gene is _ _ __ Ahhr...

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.

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