Messenger For Protein Research Articles

Upstream Flanking Sequence Assists Folding of an RNA Thermometer

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5’UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5’UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

The pregenomic RNA directs replication of the hepatitis B virus (HBV) genome by serving both as the messenger for core protein and polymerase and as the genome precursor following its packaging into the core particle. RNA packaging is mediated by a stem-loop structure present at its 5' end designated the epsilon signal, which includes the core gene initiator AUG. The precore RNA has a slightly extended 5' end to cover the entire precore region and, consequently, directs the translation of a precore/core protein, which is secreted as e antigen (HBeAg) following removal of precore-derived signal peptide and the carboxyl terminus. A naturally occurring G1862T mutation upstream of the core AUG affects the bulge of the epsilon signal and generates a "forbidden" residue at the -3 position of the signal peptide cleavage site. Transfection of this and other mutants into human hepatoma cells failed to prove their inhibition of HBeAg secretion but rather revealed great impairment of genome replication. This replication defect was associated with reduced expression of core protein and could be overcome by a G1899A covariation, or by nonsense or frameshift mutation in the precore region. All these mutations antagonized the G1862T mutation on core protein expression. Cotransfection of the G1862T mutant with a replication-deficient HBV genome that provides core protein in trans also restored genome replication. Consistent with our findings in cell culture, HBV genotype A found in African/Asian patients has T1862 and is associated with much lower viremia titers than the European subgroup of genotype A.

Coat protein enhances translational efficiency of Alfalfa mosaic virus RNAs and interacts with the eIF4G component of initiation factor eIF4F

The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.

Influence of Deep Hypothermia on the Tolerance of the Isolated Cardiomyocyte to Ischemia–Reperfusion

The influence of deep hypothermia (4°C) during a substrate-free, hypoxia–reoxygenation treatment was investigated on cardiomyocytes (CM) prepared from newborn rat heart in culture in an in vitro, substrate-free model of ischemia–reperfusion. The transmembranous potentials were recorded with standard microelectrodes. The contractions were monitored photometrically. The RNA messenger (mRNA) and protein expression for protein (HSP70) were analysed by RT-PCR (reverse transcriptase-polymerase chain reaction) and Western blotting, respectively. Simultated ischemia (SI) caused a gradual decrease and then a cessation of the spontaneous electromechanical activity. During the reoxygenation, the CM recovered normal function, provided that SI did not exceed 2.5 h. When SI duration was increased up to 4 h, reoxygenation failed to restore the spontaneous electromechanical activity. Conversely, the exposure of the CM to SI together with deep hypothermia decreased the functional alterations observed, and provided a complete electromechanical recovery after 2.5 h as well as after 4 h of SI. Deep hypothermia alone failed to induce HSP70 mRNA and protein production. On the contrary, HSP70 mRNA production increased after 2.5 and 4 h of deep hypothermia followed by 1 h of rewarming, proportionally to the duration of the cooling period. This augmentation in mRNA was associated with a rise in HSP70 protein content. In summary, it appeared that deep hypothermia exerts a strong cytoprotectrive action during SI only, whereas cooling CM before SI has no beneficial effect on subsequent SI. Moreover, these results suggested the persistence of a signaling system and/or transduction in deeply cooled, functionally depressed cells. Finally, CM in culture appeared to be a model of interest for studying heart graft protection against ischemia-reperfusion and contributed to clarifying the molecular and cellular mechanisms of deep hypothermia on myocardium.

Genome characterization and taxonomy of Plantago asiatica mosaic potexvirus

The complete nucleotide sequence of Plantago asiatica mosaic virus (P1AMV) genomic RNA has been determined. The 6128 nucleotide sequence contains five open reading frames (ORFs) coding for proteins of M(r) 156K (ORF1), 25K (ORF2), 12K (ORF3), 13K (ORF4) and 22K (ORF5). The sequences of these P1AMV proteins exhibit strong homology to the proteins of the other potexviruses. Phylogenetic trees based on the multiple sequence alignments of three conserved domains in ORF1 product and capsid protein reveal a close relationship of P1AMV to papaya mosaic virus and clover yellow mosaic virus. The P1AMV genomic RNA and a major subgenomic RNA (sgRNA) of 0.9 kb have been detected in infected leaves by Northern blot hybridization. The latter sgRNA is the messenger for virus capsid protein and its 5' terminus has been located 23 nucleotides upstream of the initiator codon of the coat protein gene. The P1AMV virion RNA and RNA transcript resembling the 0.9 kb sgRNA have been translated in vitro giving rise to a single major 170K product and a major 22K product, respectively.

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.

Biologically active transcripts of cloned DNA of the coat protein messenger of two plant viruses

To initiate infection, a mixture of the three genomic RNAs of alfalfa mosaic virus (AIMV) has to be supplemented with a small amount of coat protein or RNA 4, the subgenomic messenger for coat protein. The possibility to replace RNA 4 in the inoculum by in vitro synthesized transcripts of a cloned DNA copy of the coat protein cistron was investigated using the SP6 transcription system. Transcripts with or without the cap structure m(7)G(5')ppp(5')G were both translated in vitro in viral coat protein, but only capped transcripts yielded an infectious mixture when added to the AIMV genomic RNAs. This indicates that the cap structure is essential to the in vivo translatin of RNA 4. Similar results were obtained with RNAs transcribed in vitro from a DNA copy of the putative coat protein cistron of tobacco streak virus (TSV). re]19850822 rv]19851203 ac]19860114.

Autonomous Replication and Expression of RNA 1 from Black Beetle Virus

Black beetle virions contain two RNAs. The smaller one, RNA 2, has previously been shown to be a messenger for viral coat protein. It is shown here, by infecting sensitized Drosophila cells with the individually purified RNAs, that the larger one, RNA 1, carries the viral gene(s) required for RNA polymerase functions. RNA 2 was dispensible for synthesis of viral RNA 1 and subgenomic RNA 3 but was essential for synthesis of RNA 2 and virions. Cells infected with RNA 1 alone produced RNA 3 in proportions 10- to 20-fold greater than cells infected with virions. This overproduction of RNA 3 decreased with increasing proportions of RNA 2 in the infecting RNA 1. We conclude that RNA 1 is the previously unidentified progenitor of subgenomic RNA 3, whereas RNA 2 regulates the amount of RNA 3 produced in the infected cell.

Black Beetle Virus: Messenger for Protein B Is a Subgenomic Viral RNA

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein alpha (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected Drosophila cells. Two of these proteins, A and alpha, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and alpha cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.

Evidence that RNA 4 of alfalfa mosaic virus does not replicate autonomously

A mutant (Tb ts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30°, can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tb ts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).

3'-Terminal nucleotide sequence of alfalfa mosaic virus RNA 4.

The sequence of the 3'-terminal 91 nucleotides of alfalfa mosaic virus RNA 4, the messenger for the viral coat protein, has been elucidated. A fragment containing the 3' terminus of the RNA was obtained by mild digestion with RNase T1. The primary structure of the fragment was deduced by labeling it in vitro at its 5' terminus and application of RNA sequencing techniques. The sequence is completely extracistronic and is believed to contain the binding sites for the viral coat protein and replicase.

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.

Early Events in the Infection of Tobacco with Alfalfa Mosaic Virus

SUMMARY At 23 °C, infections with alfalfa mosaic virus can be initiated with the three genomic RNA species in combination either with coat protein or with the messenger for coat protein (RNA 4). At 30 °C the combination of the three genomic RNA species with coat protein is still infectious to tobacco, whereas the infectivity of the combination with RNA 4 is almost nil. The combination of the four RNA species is infectious to bean at both temperatures. Tobacco leaf discs inoculated with the four AMV RNA species were incubated for short periods at 30 °C followed by 4 days at 23 °C. Infectivity could only be recovered when the shift-down occurred within 10 min after inoculation. Longer exposure to 30 °C probably leads to the degradation of one or more of the AMV RNA species. Translation of RNA 4 into coat protein at 23 °C in the inoculated cells presumably will render the RNA infection resistant to 30 °C. To measure the minimum time required for the penetration and translation of RNA 4, tobacco leaf discs inoculated with the four AMV RNA species were incubated for different periods at 23 °C and then for 4 days at 30 °C. From the results it was estimated that 15 to 30 min is sufficient for the penetration and translation of RNA 4.

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.

Nucleotide sequence of a viral RNA fragment that binds to eukaryotic ribosomes.

The nucleotide sequence has been determined for the first 53 bases of brome mosaic virus RNA4, the monocistronic messenger for brome mosaic virus coat protein. The sequence includes the binding site for wheat embryo ribosomes. The 5'-terminal base is a modified guanosine attached to the penultimate base through a 5' p-p-p 5' link. The initiating AUG codon is only 10 nucleotides from the 5'-terminus. The triplets following the AUG codon correspond to the known sequence of brome mosaic virus coat protein.

Translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo.

The four RNAs of brome mosaic virus induce substantial incorporation of amino acids into protein when used as messengers in a cell-free protein-synthesizing system derived from wheat embryo. RNA 4 is highly efficient as a monocistronic messenger for the viral coat protein. Acetate, derived from acetyl coenzyme A, is incorporated into the product made in vitro. Although RNA 3 also contains the coat-protein cistron, it induces synthesis mostly of a protein larger than coat protein. RNAs 1 and 2 also induce the synthesis of substantial amounts of protein other than coat protein. However, an equimolar mixture of RNAs 3 and 4 or of 1, 2, 3, and 4 induces synthesis of coat protein almost exclusively. This result suggests that the coat-protein cistron, when present as a monocistronic messenger, inhibits translation of all other viral messages.

Juvenile hormone-controlled synthesis of female-specific protein in the cockroach Leucophaea maderae

Application of 1 μg of the all-trans methyl (±)-12,14-dihomojuvenate induces the de novo synthesis of a female-specific protein at a rate similar to that found in unoperated egg-maturing females. JH also appears to stimulate an increased synthesis of the non-sex-specific proteins. The female-specific protein is essential for egg maturation since it makes up 85–90% of the egg proteins. The exact role of the non-sex-specific proteins in egg maturation processes is not known. 12,14-Dihomojuvenate and 12-homojuvenate, the two naturally occurring JHs, have similar potencies in inducing the synthesis of female-specific protein. The 14-homojuvenate, not known to occur naturally, also has similar potency. Epoxyfarnesenic acid is 10–20 times less potent. The all-trans isomers of either of the two JHs appear to be 2–10 times more potent than the naturally occurring hormones, whereas the t,c,t-isomer of the 12,14-dihomojuvenate has no activity. The JHs possibly influence transcription of specific mRNA since application of actinomycin D (2 μg) simultaneously with JH completely blocked female-specific protein synthesis even when assayed 5 days later. Messenger for the specific female protein is relatively long lived (at least 3–4 days).

Regulation of ribosomal protein synthesis in Escherichia coli.

The kinetics of synthesis of ribosomal, nonribosomal, and total protein, and of ribosomal ribonucleic acid (RNA), were measured in Escherichia coli during a shift-up involving a doubling of specific growth rate. The increase in ribosomal protein synthesis was not accompanied by a corresponding decrease in non-ribosomal protein synthesis. Thus, the average rate of protein synthesis per ribosome increased after the shift. The increase did not occur by an increase in polypeptide chain growth rates. The average growth periods of ribosomal and nonribosomal proteins did not change after a shift, and were constant in cells in balanced growth over a wide range of specific growth rates. The average growth period for ribosomal protein was twice that for nonribosomal protein. The fraction of ribosomes in polyribosomes was found to increase after the shift, accounting for the increase in ribosomal protein synthesis and in protein synthesis per ribosome. These results show that ribosomal protein synthesis is regulated by control of initiation of either transcription or translation of ribosomal protein messenger RNA, by some means other than availability of free ribosomes. If the messenger for ribosomal protein is ribosomal RNA, the conclusions can be extended to apply to regulation of ribosomal RNA synthesis. In support of this, it is shown that ribosomal RNA and ribosomal protein synthesis are closely coordinated during a shift.