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Results for “polycistronic mRNA”

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9 recordsLinked to original sources
PubMed2025

Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5' galE-galT-galK-galM 3', via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3' end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of galE mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-galE mRNA undergo exonucleolytic processing to the 3' end of galE mRNA observed in vivo. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

RNA, Messenger↗
PubMed1976

Isolation and in vitro translation of zein messenger ribonucleic acid.

Zein messenger RNA was isolated from membrane-bound polyribosomes of developing maize kernels by oligo(dT)-cellulose chromatography. Translation of the mRNA in vitro yielded protein similar to native zein in amino acid content, ethanol solubility, and mobility on sodium dodecyl sulfate- polyacrylamide gels. The zein mRNA sedimented as a homogeneous peak on sucrose gradients and contained a poly(A)-rich region based upon hybridization to [3H]poly(U). The mRNA had an apparent molecular weight of 540 000 on agarose-acrylamide gels. It synthesized both 21 800 and 19 000 molecular weight zein components in the wheat-germ cell-free protein synthesis system. The possibility of a polycistronic mRNA or two mRNAs of similar molecular weight is considered.

Amino Acids↗
PubMed1979

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.

Animals↗
PubMed1978

Effect of RNase III on the size of bacteriophage T7 lysozyme mRNA.

The size of lysozyme mRNA from T7-infected E. coli RNase III+ and RNase III- strains was analyzed by sucrose gradient sedimentation, dimethylsulfoxide (Me2SO) sucorse gradient sedimentation, and preparative gel electrophoresis. Each technique revealed a similar size distribution of multiple lysozyme mRNA's. Analysis by preparative gel electrophoresis of RNA extracted after infection of Escherichia coli Bst (RNase III+) separated lysozyme mRNA into six peaks of activity ranging in size from 0.2 x 10(6) to 1.9 x 10(6) daltons. Four well-resolved major peaks of activity were detected, having apparent molecular weights of approximately 0.61 x 10(6), 0.76 x 10(6), 0.92 x 10(6), and 1.3 x 10(6). A broad band of activity, with a molecular weight range from 0.2 x 10(6) to 0.37 x 10(6), was also present, and a sixth peak of activity was sometimes observed that migrates with a mobility corresponding to a molecular weight of 1.9 x 10(6). Judging from their molecular weight as estimated by electrophoresis, most, if not all, of the lysozyme mRNA's were polycistronic. The RNA extracted after infection of an RNase III- host contained a more heterogeneous collection of lysozyme mRNA's. In addition to lysozyme mRNA activity on RNAs with molecular weights between 0.2 x 10(6) and 1.9 x 10(6), RNA species with molecular weights estimated at 4 x 10(6) to 5 x 10(6) were also detected. The data indicate that RNase III processes at least some of the primary lysozyme transcripts. The multiple lysozyme mRNA's represent discrete RNA species rather than aggregates because analysis of the size of lysozyme mRNA under completely denaturing conditions, in Me2SO, produced a similar size distribution of lysozyme mRNAs. Also, treatment of RNA with 90% Me2SO, which separates the strands of a completely double-stranded RNA, did not significantly alter the electrophoretic mobility of the lysozyme mRNA.

Coliphages↗
PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗
PubMed1979

On pre-messenger RNA and transcriptions. A review.

From the present review integrating old and new data emerge a few principles of gene expression in eukaryotes, and an infinite variety of possible mechanistic details generating the overal pattern. The few principles, most of which are not fundamentally new, may thus be summarized. 1) The eukaryotic genome is subdivided into transcriptional units: into transcriptons which are subject to individual activation controlled at DNA level. 2) Viral genomes may contain one or a few transcriptons, while cells of multicellular organisms contain from 3 x 10(3) in diptera up to an estimated 2 x 10(5) in birds and mammals. 3) Transcriptons may include one or several coding sequences. 4) Transcriptons vary considerably in size: in mammals and birds their size spectrum falls into the 2,000 to 20,000 bp range. 5) Units of coding information constituting one message (genes) and, possibly, units of regulative information are frequently broken up and stored within the transcripton in sub-genic blocks (of so far unknown significance) in general located at a certain distance from the 5' and 3' transcript terminals which are determined by the promotor and terminator signals. 6) The gene, in its specific definition as the functional unit underlying the phenotype, is in general constituted posttranscriptionally by the processing mechanisms from the mosaic of its genomic subunits in the transcripton; segments of coding, service and regulative sequences are recombined within themselves and with each other, polygenic transcripts separate into their unit messages. 7) Activated transcriptons produce pre-mRNA; these primary transcripts are colinear with the DNA of the transcriptional unit. 8) Primary pre-mRNA is processed into secondary pre-mRNA's by extragenic cleavage and intragenic ("splicing") processing, giving rise stepwise to functional mRNA. During this process chemical modifications as methylation, 5'-terminal capping and 3'-terminal polyadenylation take place. 9) Translation yields either potentially functional polypeptides or polycistronic polyproteins subject to further processing. 10) Processing is a regulated process; it involves many of the possible phases and mechanisms of post-transcriptional regulation (cf. 39, 40).

Animals↗
PubMed1976

Translation of T7 RNA in vitro without cleavage by RNase III.

T7 early mRNA's are generated from a high-molecular-weight precursor RNA by site-specific RNase III cleavage. When T7 DNA is transcribed in vitro by Escherichia coli RNA polymerase, the transcript is a large, single-piece RNA equivalent to the in vivo precursor RNA. The T7 RNA synthesized in vitro can be translated as a polycistronic messenger without cleavage by RNase III. All T7 early proteins are synthesized in an RNase III-free, protein-synthesizing system directed by the uncleaved T7 RNA.

Cell-Free System↗
PubMed1978

Formylation of initiator tRNA methionine in procaryotic protein synthesis: in vivo polarity in lactose operon expression.

Eucaryotic and procaryotic organisms differ in two aspects of their translation machinery: polycistronic messengers are expressed as a sequence of individual proteins only in procaryotes, and the initiation of protein synthesis proceeds with an initiator tRNA which is found to be modified (formylated) in procaryotes and not in eucaryotes. In the present study, we show that formylation is required in vivo for the coordinate expression of the Escherichia coli lactose operon. Our experiments are consistent with a translation mechanism using dissociated ribosomes at the 5' end of the mRNA in a reaction that is only weakly dependent on formylation at this initiation step; the ribosomes then travel along the messenger and can reinitiate after the intracistronic barrier without dissociation. This latter initiation step is strongly dependent on the level of formylation: a low level of the formyl group, obtained by the antifolic agent trimethoprim, induces a strong polarity in the expression of the lactose operon. There exist mutant strains in which this polarity is much less apparent than in the wild type. We show here that such is the case of rpsL mutants. Ribosomes mutated in the S12 protein (rpsL) are found to be much more easily dissociated than the wild type. This might explain why the expression of the lactose operon on rpsL strains remains coordinated when the intracellular level of formylation is decreased.

Acetyltransferases↗