David Kennell

Transcription and translation initiation frequencies of the Escherichia coli lac operon

Abstract We have calculated the number of RNA polymerase molecules transcribing the induced lac operon of Escherichia coli as well as the distance between ribosomes on the proximal z and on the distal a messages † . These values were derived from: (a) rates of induced enzyme synthesis corrected by known turnover numbers to give numbers of enzyme monomers produced per cell per second; (b) the mass of each message derived either directly by hybridization of long-labeled RNA to specific DNA or from the rates of synthesis and decay obtained by hybridization of pulse-labeled RNA; the latter gives about 13 times better resolution; (c) conversion of message mass into a number of funtioning 3′ ends (producing finished polypeptides); size analyses indicate that the z message probably decays by a net directional degradation in the 5′ to 3′ direction. From this the fraction of completed molecules that are full-length and the total number with functional 3′ ends can be derived. (d) Rates of ribosome movement. Calculated values follow. (1) With a 3.3-second interval between transcription initiations, there are 38 molecules of RNA polymerase on z DNA per cell; with 1.7 copies of lac DNA, this gives 23 on each z cistron and five or six on the y and on the a cistrons. This corresponds to a spacing of about 135 nucleotides between polymerases, which is similar to that on the ribosomal cistrons which have only 1.7 seconds separating initiations. (2) There are 20 molecules of β-galactosidase monomer produced per cell per second from 1.1 × 10−4 pg of z RNA. 38% of the message molecules are nascent and not producing enzyme. Half of the completed molecules and 70% of their mass are intact z messages to give 62 molecules of z message containing functional 3′ ends/cell. Ribosomes load onto these messages at 3.2-second intervals to give a ribosomal spacing of 110 nucleotides. An average of 40 ribosomes translate each z message with half of the message molecules translated by more than 28 ribosomes and half by less. (3) Only 2.4 molecules of galactoside acetyltransferase monomer are produced per cell per second from 1.8 × 10−5 pg of a RNA. Only 20% of these molecules are nascent and 80% of the completed ones are intact; this gives 40 molecules of functioning a messages per cell. Ribosomes load to a message at 16-second intervals to give a spacing of 580 nucleotides. These results show that the frequencies of translation initiations can differ for different messages. The faster decay of a compared to z message is consistent with a model in which a ribosome can protect a vulnerable site near the start of a message from inactivation; messages that load less frequently decay faster. The combined net effect of these causally related processes could account for natural polarity (Zabin & Fowler, 1970), i.e. there is a fourfold lower production of enzyme from the a than from the z gene.

Principles and Practices of Nucleic Acid Hybridization

Publisher Summary This chapter discusses that nucleic acid hybrid formation has been used as a research tool for approximately one decade. It has been a decade of remarkable progress toward understanding mechanisms of gene expression. Probably no other single procedure has contributed more to this success than has hybridization. However, the procedure is still usually used as a crude assay for genetic relatedness, used less often for precise quantitative estimations. Its value as a technique has become even greater with studies that are undertaken to determine the variables that contribute to final yield of hybrid. Hybridization has continued to be of great value for some time. The additional complexity of the genomes of higher organisms gives added complexity to interpretations of hybridizations with their nucleic acids. An indirect approach that is provided if small segments of eukaryotic chromosomes can be pursed, this is now possible for bacterial genes. The chapter concludes that the problems will have to be solved by more rigorous analyses and by refinements in the techniques. This will be necessary unless a chemical method more useful than hybridization is developed to study nucleic acid specificity.

Titration of the gene sites on DNA by DNA-RNA hybridization. II. The Escherichia coli chromosome.

Abstract Escherichia coli RNA, having the same specific radioactivity in all molecules (long-labeled RNA), or labeled by exposing bacteria to [ 3 H]uracil for 30 seconds (pulse-labeled RNA), was hybridized to E. coli DNA over a wide range of RNA/ DNA inputs † : from ratios so low (less than 1 1000 ) that not all copies of any DNA site were filled, to RNA/DNA inputs so high (greater than 1000) that no more RNA could be bound. RNA with radioactivity only in stable components was also hybridized over a wide range of RNA/DNA inputs. (a) The maximum fraction of input RNA detectable as authentic hybrid was about 75%; it is difficult to exceed this figure because of simultaneous formation of partial hybrids subsequently removed by ribonuclease. (b) The first DNA sites to become saturated did so at RNA/DNA inputs of about 1 1000 . All sites for stable RNA were occupied at an RNA/DNA input of about 1 160 . (c) At any RNA/DNA input between about 1 4 and 1 160 , virtually all unstable RNA is hybridized, and the size of this fraction equals the difference between the fraction of radioactivity hybridized from total RNA and from stable RNA. Such measurements suggested that unstable RNA is 2.5 to 3% of the bacterial RNA while 50% of the synthesized RNA is unstable. Competition experiments with excess unlabeled rRNA gave the same figures. (d) There is a wide variation in the numbers of molecules of different mRNA species per cell; 10% of the active genes are homologous to 70% of the mRNA mass . (e) Over the entire range of abundances, the same frequency distribution was found for the radioactivity of pulse-labeled mRNA. This suggests that variable life-times do not contribute to the abundance distribution of mRNA species. (f) Only 10% of the DNA was observed to be complementary to virtually all (more than 99.85%) of the label from both pulse-labeled or long-labeled mRNA; this suggests that in these cultures most of the potential E. coli gene sites make extremely little if any RNA.

Translation and mRNA Decay

SummaryDegradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition.These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearby ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.

Evidence for endonucleolytic attack in decay of lac messenger RNA in Escherichia coli

Abstract The size distribution of decaying messenger RNA molecules from the lactose (lac) operon of Escherichia coli has been measured. A one-minute induction period was terminated by rifampicin, then a further period was allowed for completion of the lac mRNA transcription. All incomplete lac mRNA molecules could then be identified as degradation products. RNA was labeled with [3H]-uracil and purified by procedures which produced no significant cleavage. The purified RNA was centrifuged in sucrose gradients and then each fraction was hybridized with excess φ80dlac DNA to determine the amounts of lac message as a function of size. The size distributions observed at various times have been compared to those expected according to different mechanisms of degradation. These include a 5′ to 3′ exonucleolytic degradation as opposed to mechanisms in which the primary attack is endonucleolytic. The results clearly exclude an exclusive exonucleolytic degradation. Although the number and distribution of sites subject to endonucleolytic cleavage cannot be determined, the results are consistent with a model in which there are vulnerable sites at the start of the message specifying each polypeptide chain.

Titration of the gene sites on DNA by DNA-RNA hybridization: I. Problems of measurement

Abstract Three procedures to form RNA-DNA complexes have been studied. (1) The filter method —RNA is reacted with denatured DNA immobilized on a filter; (2) the agar method —DNA is immobilized in agar; (3) the liquid method —both DNA and RNA are in solution. RNA was labeled by exposing Escherichia coli to radioactive precursors for either short (pulse-labeled) or long times (long-labeled). Hybrid formation was assayed by radioactivity forming a complex with the filter or agar. The filter method can be inaccurate at higher RNA/DNA inputs. With RNA/ DNA input constant , the fraction of RNA radioactivity that forms complex increases with RNA concentration up to sufficiently high concentrations at which fractional yields cannot be further increased. Concentration dependence is more marked with pulse-labeled than with long-labeled RNA, presumably because more of the label in the hybridized RNA is in the rarer species. The results are consistent with an interpretation based on the law of mass action. At high RNA/DNA inputs, radioactivity retained by a blank filter can be comparable to that in genuine hybrid especially with long-labeled RNA since a much smaller proportion of label forms hybrid. In the liquid method, both concentration dependency and blank filter “sticking” are much less significant. However, competition from DNA-DNA re-annealing can be limiting. To titrate DNA from uninfected E. coli , the filter method has been used at RNA/DNA inputs less than 40 and the liquid method at RNA/DNA inputs greater than 10 with good agreement at intermediate RNA/DNA inputs. In the agar method, the higher the RNA/DNA input the greater the significance of RNA trapped non-specifically to the agar. Furthermore, a large fraction of the RNA in a presumed hybrid is ribonuclease-sensitive.

Inactivation and degradation of messenger ribonucleic acid from the lactose operon of Escherichia coli

Abstract Metabolism of lac operon mRNA has been studied in Escherichia coli growing at 30 °C in amino acids-glycerol medium. Bacteria were induced for brief intervals before adding rifampicin to block further initiations of transcription. The amounts of the first (β-galactosidase) and the last (β-galactoside transacetylase) enzymes of the operon and the level of labeled lac mRNA (detected by hybridization to φ80dlac DNA) were then followed with time. The lac mRNA in polysomes was detected by hybridization of selected fractions from sucrose gradients. We favor the following conclusions. 1. (1) Functional decay or the loss of capacity to synthesize enzyme: mRNA chains are inactivated at random with a half-life of 1 minute 40 seconds. Some are attacked at the start of transcription, whereas others are inactivated only after several minutes. 2. (2) Chemical decay or the loss of mRNA: lac mRNA molecules are also degraded on a random basis as soon as transcription begins; however, the half-life for loss of molecules is 2 minutes 40 seconds. Total cell mRNA is degraded with this same half-life. However, breakdown of a lac molecule can occur in as short a time as it takes for its synthesis (less than four minutes per complete length). 3. (3) lac mRNA first appears in small polysomes, then also in larger ones and finally mostly in smaller structures again. There is no evidence that lac mRNA is ever free of ribosomes; however, our techniques could not detect very small amounts. 4. (4) Judging by the amounts of enzyme synthesized after very short intervals of induction, initiations of transcriptions may occur synchronously, but if so, they occur very frequently (about every 10 seconds). The different half-lives for inactivation and degradation suggest that more than one variable is involved in the destruction of a molecule of mRNA. Once started, chemical degradation may proceed at different rates for different molecules; alternatively, nucleases may move as fast on all molecules as does the RNA polymerase, but chains begin to be destroyed at different times after their inactivation.

Cloning and sequencing the gene encoding Escherichia coli ribonuclease I exact physical mapping using the genome library

The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined. A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al. [Cell 50 (1987) 495-508]. DNA from these clones carry almost the entire E. coli chromosome in overlapping segments. Two overlapping clones hybridized to the probe sequence. From one of them DNA containing the rna gene was subcloned and sequenced. The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A. Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme. Sites for eight restriction enzymes had been mapped on each lambda clone. By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min). This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E. coli genes.

Metabolism of messenger RNA from the gal operon of Escherichia coli

Abstract The size and metabolism of messenger RNA from the galactose ( gal ) operon of Escherichia coli has been studied. The three known structural genes of the operon are about 1100 nucleotide pairs each and code for an epimerase ( E ), transferase ( T ) and kinase ( K ) gene, in that order. (1) The full-length gal mRNA in both B and K12 strains is about 4500 nucleotides or 1200 longer than that predicted for these three genes. The transcription time to the end of the epimerase gene was estimated by the lag before appearance of transferase mRNA. These measurements are consistent with about 520 nucleotides of gal mRNA proximal to the transferase message. The position of the remaining additional nucleotides is not known. (2) The functional inactivation rate of each message was measured by following the decreasing rate of enzyme synthesis after a one-minute induction by fucose. The half-life for each message was: epimerase 1.0 minute, transferase 0.6 minute, and kinase 1.5 minutes. (3) Mass decay of gal mRNA, induced for one minute, was followed by hybridization to DNA from transducing λ gal phages. Messenger RNA from the operator-proximal half, operator-distal two-thirds or from the entire operon decays with a half-life of about 1.1 minutes. These data cannot determine whether or not mass decay of each message is proportional to its inactivation rate. (4) The size distributions of decaying gal mRNA were determined in sucrose gradients or polyacrylamide gels with time of decay. They show clearly that gal mRNA is attacked by endonucleolytic cleavage. Fragments of about 2200 nucleotide length accumulate and have the specificity of transferase-kinase mRNA. The rapid inactivation of transferase message may result from cleavage of the epimerase-transferase mRNA junction to generate these transferase-kinase pieces.

Processing Endoribonucleases and mRNA Degradation in Bacteria

Forty years have passed since the dramatic identification of mRNA, the unstable carrier of genetic information from DNA to protein (15, 48, 56). During the last decade, there have been scores of papers and reviews that assume that RNase E is the central enzyme for degradation of mRNA. (There have been hundreds of papers and many reviews on RNase E and mRNA degradation, with at least 60 just in the last 2 years. I regret the unintentional omission of worthy ones but only refer to examples in this short presentation.) It is appropriate to consider evidence for and against this conclusion since it bears on our understanding of overall pathways of metabolism. RNase E was identified in 1978 by Apirion et al. (4, 44, 96) as an endoribonuclease (endo-RNase) that catalyzed the maturation of 5S rRNA by two sequential cleavages at specific sites of the 9S RNA of Escherichia coli. About the same time, Kuwano (recently from training with Apirion) et al. isolated an unusual temperature-sensitive mutant called the ams (for altered mRNA stability) mutant (73, 107). About a decade later, it was shown that the ams mutation maps in the gene for RNase E, rne (9, 95, 99, 130). This identification contributed to the now widely held view that RNase E is the principal RNase for initiation of mRNA decay (e.g., see references 33, 34, 35, 49, 57, 87, 122, and 124).