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At least 289 records · Page 16Linked to original sources
PubMed1987

Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors.

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.

Cell Nucleus↗
PubMed1988

Messenger RNA recognition in Escherichia coli: a possible second site of interaction with 16S ribosomal RNA.

Examination of the nucleotides following the ATG or GTG initiation codons of a file of 251 genes from Escherichia coli has shown that 247 (98.4%) of them contain a sequence of at least three and 168 (66.9%) of them a sequence of at least four consecutive nucleotides that is complementary to some part of the 16 nt at the 5' terminus of the bacterial 16S rRNA. It is proposed that this sequence, which falls within the first 24 nt coding for the genetic message, might be involved in mRNA recognition through a mechanism analogous to the well-established 'Shine--Dalgarno' interaction with the 3' terminus of the 16S rRNA. Comparison of these data with data derived from a file of 117 'false' gene starts that have a Shine--Dalgarno-like sequence followed by a suitably spaced ATG or GTG triplet but which are believed not to lie at the beginnings of genetic messages shows the association that we have found to be statistically significant at the 99.9% level.

Escherichia coli↗
PubMed1977

Messenger RNA regulation in humam diploid fibroblasts.

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By camparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of of cyoloheximide.

Cell Division↗
PubMed1977

Coordinate regulation of protein synthesis and messenger RNA content during growth arrest of suspension Chinese hamster ovary cells.

We have found Chinese Hamster Ovary cells, cultured in suspension, are subject to growth control by serum. When suspended in medium containing 0.5% serum the cells becomes reversibly arrested in the beginning of the G1 phase of the cell cycle and can be maintained in this viable, nonproliferating state for several days. This system was used to examine the regulation of protein synthesis with growth rate. In particular, the experiments addressed the question whether mRNA content is the principal controlling factor determining the rate of protein synthesis. The rate of leucine incorporation in resting cells in low serum is 2-to 2.5-fold lower than that of cells growing in 10% serum. The steady-state number of cytoplasmic poly A (+) RNA molecules shows a proportional decrease, consistent with it being a determining factor controlling the rate of protein synthesis. Furthermore, the rate of production of poly A (+) and poly A (-) RNA appears to be regulated coordinately. Regulation of the rate of initiation of translation would result in fewer ribosomes bound per active message and/or a lower proportion of total mRNA's being active. Our measurements indicate that the fraction of cytoplasmic poly A (+) mRNA in polyribosomes and the relative degree of loading of each active poly A(+) mRNA with ribosomes is the same in resting and growing cells. Thus these cells resemble 3T6 and translational control does not appear to be an important part of the change in protein synthetic rate with teh state of growth.

Animals↗
PubMed1993

Leishmania pifanoi: kinetics of messenger RNA expression during amastigote to promastigote transformation in vitro.

Conditions have been developed which induce axenically grown Leishmania pifanoi amastigotes to transform into the promastigote stage in a highly reproducible fashion. Transformation was induced by a temperature shift from 31 to 22 degrees C, was inhibited by high cell concentration (> or = 40 x 10(6) cells/ml), and was unaffected by pH from 5.5-7.2. Morphologic transformation was first evident at 8 hr after induction, had occurred in > 50% of cells by 24 hr, and was > 90% complete by 48 hr. This system enabled study of the kinetics of mRNA expression during the transformation of Leishmania. The differentially expressed mRNAs for ATPase 1a and 1b, alpha- and beta-tubulin, P100/11E, Pro-1, and pLm 2, 7, 14, and 16 exhibited complex patterns of temporal expression, suggesting a highly regulated process. Differentiation on the biochemical level was evident within an hour and continued throughout the course of morphologic transformation. In addition, transformed L. pifanoi promastigotes in the plateau growth phase expressed genes characteristic of metacyclic promastigotes. Axenically cultured L. pifanoi should provide an excellent model for the study of differentiation in Leishmania.

Adenosine Triphosphatases↗
PubMed1993

Ribosome initiation complex formation with the pseudoknotted alpha operon messenger RNA.

The Escherichia coli alpha mRNA has a complex pseudoknot secondary structure that forms the recognition site for a translational repressor, ribosomal protein S4, and also encompasses the regulated ribosome binding site. To find out whether the pseudoknot is a stable structure under the conditions of ribosome initiation complex formation, thermal denaturation of the RNA was monitored by calorimetry and ultraviolet light hyperchromicity. The secondary structure formed by the coding region melts in a single transition and has a stability of -7.4 kcal/mol at 37 degrees C (5 mM-Mg2+, 100 mM-Na+, pH 7.0). A broad transition with tm approximately 38 degrees C may be a rearrangement of pseudoknot secondary or tertiary structure. Using reverse transcriptase primer extension assays ("toeprints") to measure the kinetics of ternary 30 S subunit-tRNAf(met)-alpha mRNA translational initiation complex formation, we find a fast and a slow phase in the reaction. The fraction reacting rapidly is sensitive to temperature and mutations in the mRNA. We interpret these results in terms of "active" and "inactive" mRNA conformations that are trapped by 30 S subunits and react rapidly or slowly with tRNAf(met), respectively; the active form is predominant above 37 degrees C. The binary 30 S-mRNA complex in the inactive form stops MMLV reverse transcriptase near the 3' edge of the pseudoknot structure, apparently by stabilizing the pseudoknot. We propose the following mechanism for translational initiation with the alpha mRNA. The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNAf(met). The inactive to active transition of the pseudoknot, which may be related to the 38 degrees C transition seen in melting experiments, is required for tRNAf(met) to pair with the anticodon and is rate-limiting for initiation complex formation at lower temperatures. A novel feature of this proposal is that the mRNA structure affects a kinetic step in initiation complex formation, as well as ribosome binding affinity.

Base Sequence↗
PubMed1997

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

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta 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 Qbeta 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 Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗
PubMed1993

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

Base Sequence↗
PubMed1975

The role of IF-3 in the translation of T7- and phi80trp messenger RNA.

The DNA dependent synthesis of proteins was studied with a system composed of DNA, washed ribosomes, centrifuged (150,000 X g) bacterial extract from Escherichia coli and purified initiation factors IF-1 and IF-2. Synthesis of active enzymes encoded by the tryptophan (trp)-operon of E. coli was found to depend strongly on the addition of IF-3, with the same IF-3 dependency for all 5 gene-products of this operon, irrespective of the presence of the promotor proximal gene trpE. Synthesis of T7 RNA polymerase with T7 DNA as a template, however, was completely independent of the addition of IF-3. The same difference in IF-3 requirement was found when we compared the overall protein synthesis directed by these templates. This difference could be related to the effect of IF-3 on the formation of initiation complexes with the in vitro prepared mRNA: initiation complexes are readily formed with T7 mRNA also in the absence of IF-3, whereas the formation of these complexes with phi80trp mRNA almost completely depends on the presence of this factor.

Coliphages↗
PubMed1983

Polyamines enhance readthrough of the UGA termination codon in a mammalian messenger RNA.

The polyamines spermidine and spermine stimulate the readthrough of the UGA termination codon of rabbit beta-globin mRNA when it is translated in a rabbit reticulocyte cell-free system. The other major polyamine, putrescine, does not show this effect. The polyamine induced readthrough is specific for UGA as the UAA termination codon of alpha-globin mRNA is not read through and general translational misreading errors are not occurring in the presence of spermidine or spermine. The probable mechanism of this effect and some possible regulatory implications are discussed.

Animals↗
PubMed1977

Function of the tof gene product in modifying chemical stability of trp messenger RNA synthesized from the PL promoter of lambda trp phage.

The trp operon translocated into the early region of phage lambda can be transcribed under the control of two promoters, the authentic trp promoter (Ptrptrp mRNA) and the PL promoter of the N gene (PLtrp mRNA) (Imamoto and Tani, 1972; Ihara and Imamoto, 1976a). PLtrp mRNA is stabilized with time after infection: at early times after infection chemical degradation of PLtrp mRNA is two-fold slower than for Ptrptrp mRNA, while at later times the stabilization of PLtrp mRNA is almost total. The stabilization of PLtrp mRNA is markedly reduced when the activity of the tof gene product is low due to a missense mutation of the tof gene. In contrast there is no significant reduction in stabilization when N function is lost by an amber mutation. On the basis of these and other experiments with lambdatrp susN7 tof12 phage, it is inferred that stabilization of the PLtrp mRNA is brought about by a modification of the "decay trigger", at least in part by the protein product of the tof gene.

Coliphages↗
PubMed1978

The effect of the messenger RNA concentration on the competitive inhibition of translation by cap-analogues.

Inhibition of translation of several mRNA species in a micrococcal nuclease treated reticulocyte lysate by cap analogues was compared with the competition between two mRNAs. Inhibition characteristics were very similar, only complete mRNA molecules inhibited at concentrations 150 times lower than m7 G5'ppp5'G. The inhibition of mRNA translation by cap analogues could be neutralized by the addition of extra mRNA in a manner predicted from the competitive nature of the inhibition by cap analogues.

Binding, Competitive↗