Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. 1. Ribosomal RNA chain growth rates

The chain growth rate for ribosomal RNA was determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36) and glucose/ amino acids (μ = 2.10) medium. With increasing bacterial growth rate the chain growth rate increases from 4400 to 6300 nucleotides/min. These values are almost twofold higher than the chain growth rate reported for messenger RNA; this implies that, following a nutritional shift-up, the transfer of a relatively small number of RNA polymerase molecules from unstable to stable RNA genes along with the increase in the stable RNA chain growth rate is sufficient to account for the abrupt increase in the net rate of RNA synthesis. Furthermore, our calculations indicate that the linear density of polymerase molecules on the ribosomal DNA template increases with the bacterial growth rate, such that in rapidly growing bacteria all ribosomal RNA genes (48 copies at μ = 3) are nearly saturated with RNA polymerase.     

Differential rate of ribosomal protein synthesis in Escherichia coli B-r

The differential rate of ribosomal protein synthesis, α_r (ribosomal protein synthesis rate/total protein synthesis rate), was measured for Escherichia coli strain B/r growing at different steady-state rates ranging from 0.67 to 2.3 doublings/hour. For growth rates above 1.2 doublings/hour, α_r was found to be proportional to the growth rate μ (doublings/h), such that α_r = 0.09 μ, and the ribosome efficiency (amino acids polymerized/second per ribosome), calculated from α_r, was found to be 14 to 18 amino acids/second per ribosome. With decreasing growth rates below 1.2 doublings/hour, α_r was found to be increasingly greater than 0.09 μ and the ribosome efficiency gradually decreased such that at μ = 0.67, α_r = 0.085, and the ribosome efficiency was reduced by 30% and was equal to 10 to 13 ammo acids/second per ribosome. These results imply that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 × 10⁸ to 5 × 10⁸ amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10⁸ to 4 × 10⁸ amino acids/genome. These relationships were verified by direct measurements of the amounts of DNA, RNA and protein at different steady-state growth rates. In addition, protein accumulation was measured following a nutritional shift-up from succinate to glucose minimal medium. The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up.     

Synthesis of RNA polymerase in Escherichia coli B-r growing at different rates

Polyacrylamide gel electrophoresis of unfractionated sodium dodecyl sulfate lysates of Escherichia coli$\text{B}\text{r}$ has been used to investigate the synthesis of β and β′ subunits of RNA polymerase as a function of bacterial growth rate. In succinate (μ = 0.67 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) supplemented media, the fraction of [¹⁴C]leucine-labeled β and β′ protein/total protein was found to be 1.05, 1.31 and 1.56%, respectively. Comparison of these values with recent estimates from this laboratory of the differential rate of synthesis of functioning RNA polymerase suggests an excess of total over functioning RNA polymerase. The significance of these data in reference to the regulation of RNA polymerase synthesis is discussed.     

Arabinose-leucine deletion mutants of Escherichia coli B-r

The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established.     

Metabolism of D-arabinose by Escherichia coli B-r

The pathway for d-arabinose metabolism in Escherichia coli B/r has been determined. Evidence is presented to support the following metabolic scheme: d-arabinose d-ribulose right harpoon-up d-ribulose-5-phosphate d-xylulose-5-phosphate.     

L-arabinose binding protein from Escherichia coli B-r

A protein which is capable of binding l-arabinose-1-(14)C has been isolated from l-arabinose-induced cultures of Escherichia coli B/r. Analysis for this l-arabinose-binding protein (ABP) in a number of l-arabinose-negative mutants suggests that the ABP is not coded for by any of the known genetic units of the l-arabinose complex yet is under the control of the regulator gene araC. The ABP has been purified and found to bind l-arabinose, d-fucose, d-xylose, and l-ribulose with decreasing affinities. The K(m) for l-arabinose is 5.7 x 10(-6)m. The molecular weight, as determined by equilibrium centrifugation, was found to be 32,000. The protein was observed to have many features that liken it to other recently isolated binding proteins that have been implicated in the active transport of small molecules.     

Thiolation of transfer RNA in Escherichia coli varies with growth rate.

We have used an affinity electrophoresis assay which when combined with Northern hybridization techniques permits us to estimate the degree of thiolation of individual tRNA species in Escherichia coli. We observe that the levels of 4-thio 2'(3')-uridine (4-thioU) in many but not all tRNAs varies dramatically at different bacterial growth rates: Five tRNAs are completely thiolated at all growth rates, while another eight tRNAs are incompletely thiolated and the fraction of the unthiolated form of these tRNA species increases as the growth rates increase. Transfer RNA(2Glu) contains 4-thioU as well as (methylamino)methyl-2-thio uridine (mnm(5)2-thioU). The level of mnm(5)2-thioU of tRNA(2Glu) is invariant with growth rate. Surprisingly, none of the thirteen tRNA species that we have studied is completely unmodified in all growth media. In particular, at the slowest growth rates every tRNA class that we have studied contains a form that has 4-thioU residues.     

Non-random segregation of DNA strands in Escherichia coli B-r

The segregation of DNA strands during growth of Escherichia coli$\text{B}\text{r}$ has been studied under conditions in which the chromosomal configuration and the ancestry of the cells during growth and division were known. Cells containing either one or two replicating chromosomes were pulse-labeled with [³H]thymidine, and the location of the radioactivity within chains of cells formed by growth in methylcellulose was determined by autoradiography. The locations of the radioactive cells within chains obtained after the second, third and fourth divisions were consistent with the co-segregation of only one of the replicating strands of each chromosome and a fixed region of the cell into daughter cells. The attachment of this strand to the region appeared to become permanent at the time the strand was used for the first time as a template. It is concluded that the segregation of DNA molecules into daughter cells is non-random in E. coli B/r.     

Origin and sequence of chromosome replication in Escherichia coli B-r

The initial rates of induced synthesis of tryptophanase, beta-galactosidase, and d-serine deaminase were measured in relation to the chromosome replication cycle of Escherichia coli B/r. Exponentially growing cultures were exposed briefly to (14)C-thymidine or the appropriate inducers (or both), and the amount of label or enzyme (or both) in cells of different ages was found by measuring these quantities in their progeny. The rates of induced synthesis of the three enzymes increased abruptly at about 4, 20, and 34 min, respectively, after the start of a round of replication lasting 40 min. By matching this sequence to the ind, lac, and Dsd loci on the genetic map of E. coli K-12, it was estimated that replication began at about 8 o'clock (60 min) and proceeded clockwise. In rapidly growing cells, the sequence during the division cycle was consistent with the concept that rounds of replication overlapped.     

The polypeptide chain growth rate in amino acid-starved Escherichia coli determined by a novel method

The proteins synthesized by arginine-requiring Escherichia coli during growth or arginine starvation were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate to give size distributions. The proteins made during amino acid starvation were smaller than those made by growing cells. This was true for otherwise isogenic rel⁻ (“relaxed”) and rel⁺ (“stringent”) bacteria.Also using electrophoretic profiles, the peptide chain growth rate was estimated by a novel method based on comparison of theoretically predicted and observed kinetics of pulse labeling protein chains of different sizes. During arginine starvation, the rate was 2–5 amino acids/s for both rel⁻ and rel⁺ cells, compared to 20 amino acids/s for growing cells. The results rule out chain growth-rate differences as an aspect of the “relaxed” phenomenon.