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It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations ( Fischel-Ghodsian, 1998 . Mol. Genet. Metab. 65, 97–104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified ( O'Brien et al., 1999 . J. Biol. Chem. 274, 36043–36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255–7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

Large sequencing projects require an efficient strategy to generate a series of overlapping clones. This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion. We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence. To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector. Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences. Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31. Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions. This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions.

Publisher Summary The many demonstrations that the Escherichia coli (E. coli ) rne gene product (RNase E) is involved in messenger RNA (mRNA) decay have given real impetus to the study of this unusual protein's properties and role. The recent attention given to the polyadenylylation of bacterial mRNAs and the discovery that polyadenylylation plays a role in the turnover of an E. coli plasmid-specific RNA show that bacterial mRNA decay has similarities to the eukaryotic process. Functional decay of a messenger refers to the inactivation of a messenger's template activity, whereas chemical decay refers to the degradation of the mRNA itself. Functional decay, as a process distinct from the cleavages that initiate degradation, has been demonstrated for only a few mRNAs. Specific messengers have half-lives that range from about 0.5 to 20 minutes. The rates of decay of many messengers are actively regulated, thus, affecting the yields of the proteins synthesized. Unique to bacteria, portions of polycistronic mRNAs may decay at different rates, independent of the size of the segment or its position in the transcript. Overall, the decay of mRNA in bacterial cells is a major metabolic function. The turnover of mRNA constitutes about half of the cells' RNA synthesis; the actual amount is related to their growth rate.

The scission of RNA by the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu) has been studied using a lac mRNA fragment and tRNAphe as substrates. Since the chemical mechanism of scission involves oxidative attack on the ribose, scission is observed at all nucleotides including dihydrouridine and Y-bases. Specificity for single-stranded loop regions is apparent from the similarity of the reactivity of OP-Cu to the single-strand specific reagents dimethyl sulfate and diethyl pyrocarbonate using the fragment of lac mRNA as a substrate. Similar preference is observed in the reaction with tRNA although scission in the helical acceptor stem is also observed.

Analysis of both RNA pulse-labeled in vivo with L-[Me-3H]methionine and pools of methyl-accepting RNA from normally growing Escherichia coli indicates that methylation, by and large, takes place on molecules of the same size as mature tRNA rather than on longer precursor molecules. Two forms of such methyl-accepting tRNAs have been detected. One form, co-fractionating on methylated albumin—kieselguhr columns with mature tRNA, is apparently similar in structure to mature tRNA. The other form, fractionating similarly on methylated albumin columns to transfer RNA molecules lacking the modified nucleoside pseudouridine (prepared by substituting 5-fluorouracil for uracil in tRNA), apparently lacks this modified nucleoside. Using model substrates similar to the cellular substrates described above, kinetic studies were performed both in vitro and in vivo to determine whether there is a random or an ordered formation of the individual methylated bases in E. coli tRNA. Results indicate that, with the possible exception of 2-methyladenine formation, E. coli tRNA methylating enzymes act in random sequence in catalyzing the methylation of tRNA.

Maps of genetic linkage and restriction enzyme cleavage sites can be quickly prepared on an IBM PC microcomputer with the commercially available program Lotus 1-2-3. Data can be entered on the keyboard or imported from other programs. The maps can be displayed on the screen or with a printer or plotter. These procedures should be useful in the research laboratory, in preparing figures for publication and in teaching.

The nucleotide sequence spanning the ribosomal RNA (rRNA) genes of cloned human mitochondrial DNA reveals an extremely compact genome organization wherein the putative tRNA genes are probably ‘butt-jointed’ around the two rRNA genes. The sequences of the rRNA genes are significantly homologous in some regions to eukaryotic and prokaryotic sequences, but distinctive ; the tRNA genes also have unusual nucleotide sequences. It seems that human mitochondria did not originate from recognizable relatives of present day organisms.

The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique. Purified E. coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes. The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase. Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site. A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon. In addition, an enhanced reaction occurred between the fMet codon and the distal site. These results imply an internally selective interaction between the ribosome and the mRNA sequence. The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed.

The entry of newly labeled ribosomal subunits and mRNA into polysomes was examined in the yeast mutant rna1. The entry of both types of RNA into polysomes is inhibited rapidly at the restrictive temperature. Analysis of the labeling of the ATP pool and the kinetics of synthesis and processing of mRNA at the restrictive temperature leads to the conclusion that the primary defect in the mutant affects transport of both ribosomes and messenger across the nuclear membrane.

A modification of the nondenaturing procedure of Sueoka and Hardy (1) for the isolation of transfer ribonucleic acid is described. This procedure has been used for the large scale isolation of transfer-RNA from extracts of Escherichia coli and involves a batch treatment with silicic acid, DEAE-cellulose column chromatography, and an optional ethanol precipitation of the product. The final product is homogeneous when run in polyacrylamide gel electrophoresis and highly active with respect to aminoacylation.

Alkaline hydrolyzates of RNA made in vivo by Escherichia coli contain ribonucleoside-3'-monophosphate-5'-triphosphates. These probably arise by hydrolysis of the initial nucleoside triphosphate from the 5' terminus of the nascent RNA chains. Logarithmically growing cultures, labeled for 45 seconds with (32)P-labeled phosphate, yield about 2000 molecules of labeled tetraphosphate per cell, this yield increasing only slightly with continued labeling. Only the tetraphosphates of adenosine and guanosine have been found in Escherichia coli, and these two are present in approximately equal amounts.

During infection of Escherichia coli , bacteriophage T4 directs the synthesis of at least eight transfer RNAs and of two stable RNA species of low molecular weight, of unknown function. When T4 DNA is incubated with purified RNA polymerase and the appropriate substrates, a high molecular weight RNA is produced. This RNA, on further incubation with a supernatant fraction prepared from E. coli , is cleaved to several species of RNA. These cleaved RNAs were analyzed by gel electrophoresis and fingerprint techniques, and were found to be similar or identical to those made in vivo . The fingerprint analysis of one of these, a tRNA Gly , is presented. The molecule made in vitro , except for the absence of modified bases, appears to be identical to the RNA made by T4-infected cells. Therefore, in this system the tRNA genes are transcribed with fidelity, the transcript is cleaved correctly, and the tRNAs are made in good yield.

It is well known that bacterial cells regulate their rate of RNA accumulation (net RNA synthesis) in strict accord with the potential of a particular medium to support growth at a given rate, and that in shifts from one medium to another there are rapid and seemingly preferential changes in the net rate, while the other parameters of cell growth change more slowly. In this study, in addition to making measurements of net rates of RNA synthesis in steady states and shifts from one medium to another, measurements of the total rate, that is the sum of the rates of synthesis of stable and unstable RNA species, are made. This is done by measuring the early slope of the incorporation of a radioactive precursor ([3H]guanine) into RNA, and correcting for the specific activity of the intracellular precursor ([3H]GTP) at those times. It is found that, in differing steady states of growth, cells vary, not only their total rate of RNA synthesis, but also the fraction of the total given to the formation of unstable species. Expressed as a ratio of stable to unstable RNA's, this latter value is about one to one in minimal-glucose and two to one in medium enriched with amino acids. Moreover, transients between steady states give a stronger indication that the synthesis of stable and unstable RNA species can vary independently. In shift-up for example, while the net rate of accumulation, presumably largely the synthesis of the stable ribosomal and transfer RNA's, increases immediately, giving rise to the striking increase of isotope uptake known to take place, the rate of total synthesis increases only in the course of 5 to 15 minutes, such that at early times the ratio of stable to unstable RNA's made reaches about three to one. These experiments suggest that the mechanism for the control of RNA synthesis involves both a means whereby the total capacity of the cell to make RNA is limited, as well as a mechanism by which the distribution of that capacity between template sites leading to the synthesis of stable and unstable RNA species is regulated.

EXPERIMENTS in vitro and in vivo have shown that nascent RNA chains have 5′-triphosphate termini and that the 5′ terminal nucleosides are either adenosine or guanosine1–3. Little is known about the fate of these termini. It is known that in Escherichia coli mRNA is degraded rapidly4, and that the nascent precursors of ribosomal and tRNA mature—a process involving trimming at the 5′ end5–7—and then have only one phosphate group on the 5′ end7,8. It seems, therefore, that the 5′-triphosphate termini should disappear as a result of mRNA decay and stable RNA maturation. We have examined the disappearance of triphosphate termini from E. coli RNA and found that they decay at a rate approximately one-half of that of the bulk of the unstable RNA.

Publisher Summary This chapter describes the purification procedure for phosphoribosylglycinamide synthetase from Aerobacter aerogenes organism. Phosphoribosylamine: glycine ligase [ADP] (PRG) synthetase catalyzes in the de novo path of purine biosynthesis in bacteria as well as higher organisms. The chapter discusses the preparation procedure from Aerobacter aerogenes or, according to the current classification of the original strain, Klebsiella pneurnoniae. Two assays are described. The colorimetric assay is recommended for enzyme purification. It is fast, sensitive, and works well with crude enzyme preparations. The second––radioisotopic assay––is more readily quantitated. It does not work well with crude enzyme preparations. The measurement is carried out in two steps: the formation of PRG and the PRG assay. The purification is carried out at 3° unless otherwise stated. Crude cell extracts are stable for one day of storage on ice, and enzyme at the intermediate stages of purification is stable for several days.

These experiments investigate two aspects of RNA synthesis in Escherichia coli ML30 during the transition from a relatively slow rate of growth to a more rapid one: (1) the number of growing RNA molecules per cell, and (2) the average time required for addition of a nucleotide onto a growing RNA chain. Cells were grown at 30°C in a glucose-minimal salts medium and shifted-up by the addition of Casamino acids. Measurements were made of the rates of incorporation over short intervals (e.g. 5,8,12, and 16 s) of [3H]guanine into the internal and 3′-terminal nucleotides of RNA. After correction for the specific activities of the intracellular GTP pools, and for the rate of [3H] guanine accumulation at the 3′-terminus of non-growing RNA, the rates of chain elongation were calculated. It was found that cells growing at a rate of 0.9 generations/h contain approx. 4800 RNA molecules, growing at a rate of 28 nucleotides/s per chain. Cells growing exponentially at the postshift-up rate (1.2 generations/h) contain 7000 RNA molecules per unit equivalent cell mass, which are growing at a rate of 32 nucleotides/s per molecule. Three min after shift-up, cells contain the same number or slightly fewer (10%) growing RNA molecules than cells prior to shift-up, 4300, and these are being elongated at a rate of about 32 nucleotides per s. The results are consistent with the view that in the range of growth rates studied, the total rate of RNA synthesis is regulated through a limitation in the number of functioning RNA polymerase molecules, each working at a relatively constant, presumably maximal, average rate.

Bacteriophage T4 possesses a cluster of tRNA genes which are expressed as immediate early functions of phage infection. In vitro the tRNAs appear transcribed polycistronically from a single promoter. Transcription from this site is made resistant to rifampicin inactivation by the addition with rifampicin of ATP, UTP and CTP. This stabilization, which is a common characteristic of several of the T4 early promoters, allows one to separate the initiation and elongation steps of gene expression and facilitates the study of promoter function. The tRNA genes appear to be preferentially transcribed among those operons initiated in the presence of ATP, UTP and CTP. We have studied initiation using a multistep procedure in which we assume only 1 polymerase per ATP initiated operon becomes rifampicin resistant. We find that the RNA polymerase concentration needed to support a half-maximal tRNA synthesis is 5 fold lower than that needed to support half-maximal total RNA synthesis. This difference appears to be due to differences in the affinities of the promoters for polymerase. The temperature of activation of the tRNA promoter is similar to or only slightly lower than that of other ATP initiated promoters, that is, about 12° at 0.05 M KC1 concentration. This suggests that the difference does not lie in the ease in which the open complex is formed, but rather in the recognition of the promoter site itself.

WE have determined the half life of Saccharomyces cerevisiae messenger RNA (mRNA) and its associated poly (A) sequences, from the kinetics of their labelling with 14C-adenine. Corrections have been made for changes in the ATP pool specific activity during the labelling period. This method of determining the mRNA half life is preferred to observing the decay of mRNA in the presence of inhibitors because changes in the cell metabolism caused by an inhibitor may also change the mRNA half life1. One of our purposes in determining these half lives is for their use in the characterisation of temperature-sensitive mutants which are defective in macromolecular synthesis2. For this reason we have determined the half lives of mRNA and poly(A) in two different conditions, at 23 and 36 °C.

Analysis of an almost complete mammalian mitochondrial DNA sequence has identified 23 possible tRNA genes and we speculate here that these are sufficient to translate all the codons of the mitochondrial genetic code. This number is much smaller than the minimum of 31 required by the wobble hypothesis. For each of the eight genetic code boxes with four codons for one amino acid we find a single specific tRNA gene with T in the first (wobble) position of the anticodon. We suggest that these tRNAs with U in the wobble position can recognize all four codons in these genetic code boxes either by a "two out of three" base interaction or by U.N wobble.

The kinetics of entry of newly synthesized 40 S and 60 S ribosomal subunits into yeast polysomes is described. The entry times for 40 S and 60 S subunits were found to be 3 and 8 min, respectively. The kinetics of entry of 40 S subunits into large polysomes is found to be different from the kinetics of entry of 60 S subunits into large polysomes.

The human mitochondrial (mt) genome consists of a closed circular duplex DNA approximately 10 x 106 daltons and has been the most intensely studied animal mt genetic system. The positions of the origin of replication of H strand synthesis (Crews et al. 1979), the 12S and 16S ribosomal RNA genes (Robberson et al. 1972) and 19 tRNA genes (Angerer et al. 1976) have been located on the genetic map shown in Figure 1. A number of discrete products of mitochondrial protein synthesis have been demonstrated and three of them identified as subunits 1, 2 and 3 of the cytochrome oxidase complex (Hare et al. 1980). In comparison with other mito-systems, genes for up to four subunits of the ATPase complex, one of the cytochrome bc1 complex and possibly for a ribosomal protein would be expected to be present (see review by Borst 1977). Both strands are thought to be completely transcribed symmetrically from a point near the origin of the H strand synthesis (Aloni and Attardi 1971; Murphy et al. 1975). These transcripts are then processed to give the rRNAs, the tRNAs and a number of polyadenylated but not capped mRNAs (Attardi et al. 1979). Both the L and H strands have been shown to be coding with the L strand containing the sense sequence of the rRNA genes, most of the tRNA genes and most of the stable polyadenylated mRNAs.

The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.

From Eco Rl restriction fragments of T4 DNA, several fragments have been purified to apparent homogeneity, each of which contains the cluster of eight tRNA genes carried by the bacteriophage. Under our conditions these fragments are approximately 10 fold less active on a molar basis in the in vitro synthesis of the tRNAs than the non-restricted template, yet they appear as normal templates in other respects, e.g., the synthesis of tRNAs from proximal and distal regions of the cluster. In this report the possibility that this apparent low template efficiency may be due to an adversely high RNA polymerase/DNA ratio in the reactions was examined in experiments with intact T4 DNA. An inhibitory effect of RNA polymerase was seen acting on the elongation reaction of transcription. This inhibition appears to be due to the binding of polymerase to the template. It can be reversed by the addition of poly rI or heparin to the mixtures following the initiation of transcription. At appropriately low levels of RNA polymerase, the isolated fragments are transcribed selectively. The product formed contains up to six large asymmetric species indicating that the fragments bear at least one discrete initiation site for the tRNA operon.

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32 P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not known.

Wheni a strinigent strain of Escherichia coli is starved of aii amirno acid, RNA synthesis, as usually measured, as well as protein synthesis, stops.' Several explanations have been proposed for this observation. It has been suggested (1) that the discharged transfer RNA, accumulating during amino acid starvation, inhibits the RNA polymerase;1 2 (2) that traniscriptioni anld translation are coupled, through a mechanism in which the ribosomes in the process of protein synthesis pull RNA off the I)NA template;'-' and (3) that the synthesis of one of the substrates for RNA synthesis, UTP, is subject to amino acid control.6 All these theories also reflect the genieral belief that the synthesis of all species of RNA, messenger, transfer, and ribosomal, are equally subject to the same conltrol. However, clear support for or againist this theory of "coordiniate control" has beeni lackinig due to difliculty in obtaining an unambiguous measure of the amount of messenger RNA or unistable RNA made under conditions of amino acid starvation;7 it is to this questioni that this work is addressed. In previous work it has been established that unider conditionis where there is little net RNA synthesis, as is the case during amino acid starvationl, the entry of a radioactive base into the initracellular nucleotide pool is severely restricted.'0 It is therefore possible that unider these coniditions the synthesis of unstable forms of RNA, not contributing to net RNA sytithesis, may go undetected due to the failure of the labeled precursors to eniter the intracellular pools.8' 10 That this is in fact the case, ancl that colisiderable RNA synithesis can be measured during amino acid limitation, is shown in this paper. Mllethods.-The bacterial strains uded ill this study were

Exogenously supplied radioactive uracil (or guanine) enters the intracellular pools of RNA precursors in Escherichia coli only as nucleotides are removed from these pools by net synthesis of RNA. Consequently uptake of uracil over a short period does not measure the sum of the synthesis of all forms of RNA, unstable and stable, as is often supposed. Uptake of uracil during changing conditions of growth may be influenced by changes in types of RNA's being made; under such conditions that no stable RNA is being made, the synthesis of unstable forms may be greatly underestimated.

A study has been made of the kinetics of flow of [ 14 C]uracil through the intracellular pools of pyrimidine ribonucleotides of Bacillus subtilis . The rate at which these pools fill is slow as compared to that predicted from the measured sizes of these pools and the rate of nucleic acid synthesis. These results may be a consequence of the continued entry of uracil derivatives into the pools from messenger RNA turnover, or alternatively due to an internal compartmentalization of the pool components. The uracil derivatives (UMP, UDP, UTP, UDPG), which make up about 75% of the total pool size, are rapidly equilibrated with one another; this is seen in the fact that one can only trace a sequential flow of radioactive uracil through them in experiments at lowered temperatures. On the other hand, the kinetics of uracil flow through the soluble pools is the same as that through messenger, which is consistent with the equilibration of the two through messenger turnover and recycling of isotope from the pools into the messenger. By measuring the rate of [ 14 C]uracil entry into the pools, and the size of the pools, an estimate of the size of that fraction of the RNA of the cells that is turning over is obtained (7%), which is in agreement with the value obtained with the aid of actinomycin D.

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance, and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo1 or in the proper conditions in vitro2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine4. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P -ribosyl- PP amidotransferase (I), P -ribosyl glycinamide synthetase (II), P -ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured. Using as a reference the levels of enzymes found in ghe wild type bacteria grown in unsupplemented medium, the results indicate that all of the enzymes are repressed by excess purine, and derepressed by purine starvation. However, these controls are not coordinate throughout the pathway, since each segment of the pathway shows a unique response, apparently to accommodate the special functions of the enzymes within it. The levels of three early enzymes of the pathway are primarily responsive to adenine supplementation and therefore it is concluded that these are controlled by the intracellular levels of adenine nucleotides, or perhaps those of the inosine (produced in cultures by deamination). Two of these (I and IV) are coordinately controlled, while the levels of the third enzyme (III) vary in parallel with the others, but with changes of a smaller magnitude. One enzyme (VIII-IIA) was shown to be subject to a dual or multivalent control, apparently being regulated both by the supply of adenine and guanine nucleotides. This enzyme has a dual function, catalyzing one step in IMP synthesis, and another in the conversion of IMP to AMP. Strikingly a subsequent enzyme in IMP formation, IMP cyclohydrolase, has been shown by others to be primarily controlled by guanine. In addition to its above mentioned function, this latter enzyme can be envisioned as necessary for an interconversion reaction in the pathway from adenine to guanine nucleotides. The synthesis of the enzymes studied subsequent to IMP formation, in the branches leading to AMP (IA) and GMP (IG), appear primarily controlled by their respective endproducts alone. However, the possibility that the levels of the first enzyme of GMP formation (IG) might be increased by the presence of substrate, as well as repressed by endproduct, was discussed.

In studying the synthesis of macromolecules it has been of great importance to establish a system where the biosynthetic process is carried out in a cell-free extract. This approach makes it possible to interfere with various steps in the process and to isolate the intermediates.