Polycistronic Messenger RNA Research Articles

Noncontiguous operon atlas for the Staphylococcus aureus genome.

Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome. We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin-antitoxin systems, and control of the phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes.

Endogenous miRNA-Based Innate-Immunity against SARS-CoV-2 Invasion of the Brain.

The severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, possesses an unusually large positive-sense, single-stranded viral RNA (ssvRNA) genome of about ~29,903 nucleotides (nt). In many respects, this ssvRNA resembles a very large, polycistronic messenger RNA (mRNA) possessing a 5'-methyl cap (m7GpppN), a 3'- and 5'-untranslated region (3'-UTR, 5'-UTR), and a poly-adenylated (poly-A+) tail. As such, the SARS-CoV-2 ssvRNA is susceptible to targeting by small non-coding RNA (sncRNA) and/or microRNA (miRNA), as well as neutralization and/or inhibition of its infectivity via the human body's natural complement of about ~2650 miRNA species. Depending on host cell and tissue type, in silico analysis, RNA sequencing, and molecular-genetic investigations indicate that, remarkably, almost every single human miRNA has the potential to interact with the primary sequence of SARS-CoV-2 ssvRNA. Individual human variation in host miRNA abundance, speciation, and complexity among different human populations and additional variability in the cell and tissue distribution of the SARS-CoV-2 angiotensin converting enzyme-2 (ACE2) receptor (ACE2R) appear to further contribute to the molecular-genetic basis for the wide variation in individual host cell and tissue susceptibility to COVID-19 infection. In this paper, we review recently described aspects of the miRNA and ssvRNA ribonucleotide sequence structure in this highly evolved miRNA-ssvRNA recognition and signaling system and, for the first time, report the most abundant miRNAs in the control superior temporal lobe neocortex (STLN), an anatomical area involved in cognition and targeted by both SARS-CoV-2 invasion and Alzheimer's disease (AD). We further evaluate important factors involving the neurotropic nature of SARS-CoV-2 and miRNAs and ACE2R distribution in the STLN that modulate significant functional deficits in the brain and CNS associated with SARS-CoV-2 infection and COVID-19's long-term neurological effects.

The Dmp8-Dmp18 bicistron messenger RNA enables unusual translation during cellular stress.

Alternatives to the cap mechanism in translation are often used by viruses and cells to allow them to synthesize proteins in events of stress and viral infection. In Drosophila there are hundreds of polycistronic messenger RNA (mRNA), and various mechanisms are known to achieve this. However, proteins in a same mRNA often work in the same cellular mechanism, this is not the case for Drosophila's Swc6/p18Hamlet homolog Dmp18, part of the SWR1 chromatin remodeling complex, who is encoded in a bicistronic mRNA next to Dmp8 (Dmp8-Dmp18 transcript), a structural component of transcription factor TFIIH. The organization of these two genes as a bicistron is conserved in all arthropods, however the length of the intercistronic sequence varies from more than 90 to 2 bases, suggesting an unusual translation mechanism for the second open reading frame. We found that even though translation of Dmp18 occurs independently from that of Dmp8, it is necessary for Dmp18 to be in that conformation to allow its correct translation during cellular stress caused by damage via heat-shock and UV radiation.

Efficient Plastid Transformation in Arabidopsis.

Plastid transformation is routine in tobacco (Nicotiana tabacum) but 100-fold less frequent in Arabidopsis (Arabidopsis thaliana), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.

The AlfpCre mouse revisited: Evidence for liver steatosis related to growth hormone deficiency

With great interest we read the article by Mueller et al. on the development of steatosis and hepatocellular carcinoma in mice by disrupting hepatic growth hormone (GH) and glucocorticoid receptor signaling.1 They demonstrate that abrogation of hepatic signal transducer and activator of transcription 5 (Stat5) signaling caused liver steatosis by up-regulation of genes involved in lipid uptake and synthesis. However, we have concerns regarding the use of AlfpCre+ transgenic mice to inactivate Stat5 in the liver. While generating the AlfpCre transgene, the entire human GH (hGH) gene was inserted for its introns and polyadenylation signal.2 As a consequence, a polycistronic messenger RNA (mRNA) transcript encoding Cre Recombinase and hGH was transcribed in the liver, hypothalamus, and pituitary (data not shown). However, hGH was only translated in the hypothalamus and pituitary (Fig. 1A). It has previously been described that targeted expression of hGH in the hypothalamus reduces expression of hypothalamic GH releasing hormone (GHRH), leading to GH deficiency (GHD).3 This is also the case in AlfpCre+ mice in a C57Bl6/J background, as shown by quantitative real-time polymerase chain reaction (qRT-PCR). In the hypothalamus, expression levels of GHRH were reduced by 70% (P < 0.01) and mouse GH messenger RNA (mRNA) in the adenopituitary by 61% (P < 0.05). AlfpCre+ mice also showed hypoplasia of the pituitary (Fig. 1B). Hepatic insulin-like growth factor 1 (Igf1) mRNA and Igf1 plasma protein levels were decreased by 71% (P < 0.001) and 60% (P < 0.001), respectively, and reduced phosphorylation of Stat5 in AlfpCre+ livers was shown by western blotting (Fig. 1C), confirming GHD in AlfpCre+ mice. Consequently, AlfpCre+ mice exhibit postnatal growth retardation (Fig. 1D), a finding that was also observed in AlfpCre+/−c-JunF/F mice.4 Downstream of the impaired GH signaling, AlfpCre+ livers showed increased levels of triglycerides (TGs), free fatty acids (FFAs), and cholesterol, indicating liver steatosis (Fig. 1E). Finally, genes involved in lipid uptake and synthesis, including CD36, very low-density lipoprotein receptor, and peroxisome proliferator-activated receptor gamma, were significantly up-regulated by 2.52- (P < 0.01), 5.44- (P < 0.01), and 2.09-fold (P < 0.001), respectively, an expression profile indicating liver steatosis (Fig. 1F). Altogether, AlfpCre+ mice show liver steatosis related to GHD, and therefore care should be taken to use the right controls (AlfpCre+ unfloxed mice instead of AlfpCre− floxed mice) while conditionally inactivating or overexpressing genes in livers of mice. Vincent P.E.G. Pruniau, M.Sc. Els Louagie, M.Sc., Ph.D. Bas Brouwers, M.Sc. Jeroen Declercq, M.Sc., Ph.D. John W.M. Creemers, Ph.D. Laboratory for Biochemical Neuroendocrinology Department for Human Genetics KU Leuven Leuven, Belgium Els Louagie is currently affiliated with the Laboratory for Molecular Immunology and Inflammation Department of Rheumatology Ghent University Hospital Ghent, Belgium

Expression of a polycistronic messenger RNA involved in antibiotic production in an rnc mutant of Streptomyces coelicolor

RNase III is a double strand specific endoribonuclease that is involved in the regulation of gene expression in bacteria. In Streptomyces coelicolor, an RNase III (rnc) null mutant manifests decreased ability to synthesize antibiotics, suggesting that RNase III globally regulates antibiotic production in that species. As RNase III is involved in the processing of ribosomal RNAs in S. coelicolor and other bacteria, an alternative explanation for the effects of the rnc mutation on antibiotic production would involve the formation of defective ribosomes in the absence of RNase III. Those ribosomes might be unable to translate the long polycistronic messenger RNAs known to be produced by operons containing genes for antibiotic production. To examine this possibility, we have constructed a reporter plasmid whose insert encodes an operon derived from the actinorhodin cluster of S. coelicolor. We show that an rnc null mutant of S. coelicolor is capable of translating the polycistronic message transcribed from the operon. We show further that RNA species with the mobilities expected for mature 16S and 23S ribosomal RNAs are produced in the rnc mutant even though the mutant contains higher levels of the 30S rRNA precursor than the wild-type strain.

In polycistronic Q RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA.

Sugar Receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

A ubiquitin‐based vector for the co‐ordinated synthesis of multiple proteins in plants

The genetic engineering of complex traits into crop plants will ultimately require strategies to co-express more than one protein at the same time. Here, we report the development of a ubiquitin (Ub)-based expression method that can generate two proteins from a single transcript. It contains coding regions for the proteins of interest, separated in-frame by the coding region for the C-terminal end of Ub followed by a full-length Ub. On expression in tobacco, this polycistronic messenger RNA (mRNA) is translated to produce a chimeric protein that is rapidly processed by endogenous deubiquitinating proteases to release the two proteins plus a Ub moiety in intact forms. The C-terminal protein domain is released without additional amino acids, whereas the N-terminal protein domain retains the short C-terminal end of Ub. The analysis of vectors with progressively shorter C-terminal ends indicates that only the last six C-terminal amino acids of the proximal Ub domain are needed for efficient processing in plants. By comparing the levels of luciferase and beta-glucuronidase simultaneously expressed by this method in multiple independent tobacco transformants, we synthesized consistently similar ratios of the two proteins over a wide range of protein amounts. Ub-based polyprotein vectors should facilitate the genetic engineering of crops by providing a simple method for the co-ordinated and stoichiometric synthesis of two or more proteins.

Translation by Entrapment

MOLECULAR BIOLOGY A group of relatively obscure plant viruses captured the attention of molecular biologists some 30 years ago when it was discovered that their RNA genomes contain a 3'-terminal transfer RNA (tRNA)-like structure (TLS) that can be aminoacylated. Work by Barends et al . now reveals a fascinating role for tRNA mimicry in the viral life cycle. Studying turnip yellow mosaic virus, whose proteins are encoded within a single polycistronic messenger RNA (mRNA), they find that the TLS appears to entrap ribosomes in a manner that promotes internal initiation of protein synthesis on the mRNA. This yields a viral RNA replication polyprotein, which incorporates the TLS-bound amino acid (valine) at its N-terminus instead of the customary methionine. From the perspective of the virus, changing the efficiency of this internal initiation mechanism would not only affect the relative levels of the viral proteins, but also alter the balance between viral mRNA translation, RNA replication, and virion assembly. In a broader sense, this finding raises the intriguing possibility that the TLS is a molecular fossil of an ancient RNA world in which chimeric RNAs with properties of both tRNA and mRNA functioned as protoribosomes. — PAK Cell 112 , 123 (2003) .

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another

We present evidence for translational activation of the Qβ coliphage maturation cistron, mediated by the presence of Qβ replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qβ maturation gene remains translationally “off” by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched “on” when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qβ replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Nucleotide sequence and translation of satellite tobacco mosaic virus RNA

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 × 10 6 M r ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [γ- 32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5′ termini of encapsidated STMV RNAs were not phosphorylated. The 240 3′-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5′-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5′-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5′-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 M r protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 M r) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 M r protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 M r protein was not. Time course analysis of in vitro translation demonstrated that the 6800 M r protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 M r protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5′ and 3′ termini, respectively. Nonphosphorylated 5′ termini, the degree of similarity to the 3′ terminus of two of its helper viruses, the genome organization, and the ability to function as a polycistronic mRNA are unique features for the genome of this satellite virus.

Rho-dependent transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12.

It has been generally accepted that transcriptional polarity in prokaryotic systems is due to an uncoupling of translation and transcription which unmasks latent rho-dependent termination sites in a polycistronic messenger RNA. In this report, we identify and characterize rho-dependent termination sites responsible for transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12. The ilvG gene in the wild-type E. coli K12 ilvGMEDA operon contains a frameshift site which results in termination of translation in the middle of the gene. Mutations have been characterized which restore the reading frame of this gene. In addition to allowing full-length expression of the ilvG product, these mutations cause a 3-4-fold elevation in the expression of the operon distal genes. This transcriptional polarity effect on operon distal genes also has been shown to be relieved by rho suppressor mutations. We have used in vitro transcription experiments to identify rho-dependent transcriptional termination sites downstream of the frameshift site in the ilvG gene. Three tandem rho-dependent sites have been located in the ilv'GM' gene region using transcription reactions containing linear or supercoiled plasmid DNA templates. Accumulatively, these rho-dependent termination sites account for about 80% in vitro transcription termination, which is in agreement with the in vivo measurements of transcriptional polarity on operon distal gene expression. These transcriptional experiments provide in vitro confirmation for the latent rho-dependent termination site model of transcriptional polarity.

Identification of the sequence content of four polycistronic transcripts synthesized in Newcastle disease virus infected cells

During infection, the Newcastle disease virus (NDV) genome is transcribed to produce 5 to 7 species of polycistronic messenger RNA (Wilde and Morrison J. Virol. 51, 71–76) in addition to the well characterized monocistronic messenger RNA. To identify the specific sequences present in each of the polycistronic RNA species, cDNA clones generated by reverse transcription of NDV mRNAs were characterized and used as probes on Northern blots of total NDV cytoplasmic RNA. By this method it was shown that four of these large RNA species are polycistronic transcripts containing sequences from two genes: one species contains nucleocapsid protein (NP) and phosphoprotein (P) gene sequences; another, P and membrane protein (M) gene sequences; another, M and fusion protein (F 0) gene sequences; and another, F 0 and hemagglutinin-neuraminidase protein (HN) gene sequences. The existence of these transcripts yields a transcription map order of NP, P, M, f 0, HN. The remaining RNA bands may be composed of at least three different polycistronic transcripts, each of which represents transcription through three adjacent genes.

Pertussis toxin gene: nucleotide sequence and genetic organization.

The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects. Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced. All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1, S2, S4, S5, and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.

High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA

Bacteriophage M13 gene II has been cloned in the plasmid expression vector pING1 and thereby placed under the control of the inducible araB promoter of Salmonella typhimurium. Upon induction with arabinose, gene II is transcribed as part of a polycistronic messenger RNA which initiates at the araB promoter. Subsequent translation of this message results in the coordinate, high-level expression of several proteins, including the gene II protein. Using this expression system, we have been able to overproduce gene II protein to a level of almost 15% of the total protein in Escherichia coli cells, thus providing an abundant source for its purification.

Insertion of IS1 into the rpsE gene for ribosomal protein S5 causes cold-sensitivity in Escherichia coli.

The nucleotide sequence of the rpsE gene for the ribosomal protein S5 of a mutant of E. coli showing cold-sensitive growth revealed the presence of an insertion sequence, IS1, near the 3'-end of the gene. This mutant grows very slowly even at the "permissive temperature" (30 degrees C). At temperatures below 20 degrees C, its growth becomes negligible. It is likely that the presence of IS1 disturbs the continued translation of the polycistronic messenger RNA of the "spc-operon" from the point of IS1 insertion downwards, especially at the unfavorable temperatures.

A cis-dominant regulatory mutation linked to the argB-argC gene cluster in Saccharomyces cerevisiae

The argB-argC gene cluster of Saccharomyces cerevisiae, a genetic unit of coordinate expression, is presented as a good tool for investigating the possibility of eucaryotic cells producing more than a single polypeptide directly from one polycistronic messenger RNA. This conclusion is based on the following. The argB-argC gene cluster is transcribed as a single messenger RNA molecule, starting from the argB end (Minet et al., 1979). New data are presented supporting the idea that two separate proteins, N-acetylglutamate kinase and N-acetylglutamylphosphate reductase, are produced in vivo from the argB-argC gene cluster. Further evidence is provided that the low levels of both enzyme activities in arginine-grown cells are due to repression rather than to inactivation of the enzymes. A regulatory mutation has been isolated which specifically affects the expression of the argB-argC gene cluster. It is tightly linked to the cluster and close to the argB end, the putative origin of transcription. It is cis-dominant in heterozygous diploids. It does not qualitatively affect the affinity constants of N-acetylglutamate kinase and N-acetylglutamylphosphate reductase for their respective main substrates. Additional characteristics are described showing that the mutation most probably affects an operator or attenuator site, or any other regulatory site with analogous properties controlling arginine repression on the argB-argC gene cluster. The data are consistent with several interpretations. 1. (1) The argB-argC gene cluster of S. cerevisiae is translated into a single polypeptide and subsequently processed in vivo into two distinct proteins by a specialized cutting mechanism. 2. (2) The cluster is organized as a bacterial operon. 3. (3) The dicistronic mRNA corresponding to the cluster is processed into two pieces, each of which is read independently.

Cell free synthesis of bacteriophage lambda replication proteins

Bacteriophage lambda codes for two functions directly involved in the replication for its DNA: the products of genes 0 and P [l] . These two genes are transcribed to give a single polycistronic messenger RNA [2] . A third essential element, the origin of replication, is thought to be a structure on the DNA in the region where replication of the bacteriophage chromosome starts [3]. In conjunction with host factors, the replication proteins are believed to activate the origin and lead to initiation of the DNA replication. Using an in vitro complementation system it has been possible to obtain protein fractions displaying P-activity [4] . With the O-gene product the same has not been achieved. The O-activity is known to have a short half-life in vivo [5] , whereas the P-activity is more stable. This fact can be explained in terms of a requirement of different amounts of the two proteins in the cell, different rates of their synthesis (i.e., translational control), an inherent instability of the O-protein, or a combination of these factors. We have set out to synthesize the two X replication proteins in vitro using a DNA-dependent cell free protein synthesizing system [6] . Employing DNA from wild type and amber mutant h phage we will show that under these conditions the O-protein is synthesized in sizeable amounts while the P-protein is hardly detectable. This suggests an instability of the O-protein as a reason for the rapid decay of its activity in the cell and translational control of the synthesis of the two replication proteins.

Decay of messenger RNA from the tryptophan operon of Escherichia coli as a function of growth temperature

The temperature coefficients for functional decay and chemical loss of the proximal and distal parts of the polycistronic messenger RNA of the trp operon are similar in the range of growth temperatures between 10 ° and 37 °C.