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PubMed1997

Separate elements in the 3' untranslated region of the mouse protamine 1 mRNA regulate translational repression and activation during murine spermatogenesis.

The mouse protamine mRNAs, Prm-1 and Prm-2, are translationally repressed for several days during male germ cell differentiation. The translational delay of mouse Prm-1 mRNA has previously been shown to be dependent upon cis-acting elements that reside in the last 62 nucleotides of the Prm-1 3' untranslated region (3' UTR). We have previously identified a 48/50-kDa protein that binds the 3' UTRs of both Prm-1 and Prm-2 mRNAs in a sequence-specific manner, is present in cytoplasmic fractions of postmeiotic round spermatids where the protamine mRNAs are translationally silent, and is markedly reduced in elongated spermatids where the protamine mRNAs become activated for translation. Surprisingly, the binding site for this activity maps to a region of the Prm-1 3' UTR not contained within the functional 62 nucleotides described above. In this report we show that the binding site for the 48/50-kDa protein can also delay translation of a reporter RNA in vivo, suggesting that the 48/50-kDa protein can repress the translation of Prm-1 mRNA during murine spermatogenesis. This observation proves that two separate regions of the Prm-1 3' UTR are sufficient to repress Prm-1 translation. In addition, immunocytochemistry and polysome analysis have revealed that this transgenic reporter mRNA fails to undergo proper translational activation. These results suggest that an additional region of the Prm-1 3' UTR is required for proper translational activation and that Prm-1 translational repression elements can be separated from those involved in translational activation.

Animals↗

PubMed1997

Using GCN4 as a reporter of eIF2 alpha phosphorylation and translational regulation in yeast.

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the alpha subunit of the translation initiation factor elF2, which causes an inhibition of general translation. In contrast, in the yeast Saccharomyces cerevisiae phosphorylation of elF2 alpha on serine-51 by the GCN2 protein kinase mediates the translational induction of GCN4 expression. The unique structure of the GCN4 mRNA makes GCN4 expression especially sensitive to elF2 alpha phosphorylation, and the simple microbiological tests developed in yeast to analyze GCN4 expression serve as good reporters of elF2 alpha phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian elF2 alpha kinases will functionally substitute for GCN2. Structure-function analyses of translation factors or translational regulators can also be performed by assaying for effects on general and GCN4-specific translation. Three tests can be used to study elF2 alpha phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, while GCN4 expression can be analyzed using sensitive replicaplating tests. Second, GCN4 translation can be quantitated by measuring expression from GCN4-lacZ reporter constructs. Finally, isoelectric focusing gels can be used to directly monitor in vivo phosphorylation of elF2 alpha in yeast.

DNA-Binding Proteins↗

PubMed1998

mRNA translation in yeast during entry into stationary phase.

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5'-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5'-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2alpha, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions.

Artificial Gene Fusion↗

PubMed1985

The relative translation efficiencies of reovirus messenger RNAs.

The relative translation efficiencies of reovirus messenger RNAs were measured by infecting BHK cells with serotype 1, 2, or 3 virus and measuring the rate of formation of each viral protein species and the amount of each viral single-stranded (ss) RNA species present in the infected cells throughout the multiplication cycle. The translation efficiency of each ssRNA species was then calculated as a percentage of the species that was translated most efficiently. The relative translation efficiencies of the various ssRNA species did not change significantly during the multiplication cycle and cognate mRNA species of serotype 1, 2, and 3 virus were generally translated with similar efficiencies. However, the relative translation efficiencies of the individual ssRNA species differed greatly. The most efficiently translated ssRNA species was usually species s4, followed by species m2 which, on average, was translated about two-thirds as efficiently as species s4. Species s2 and s3 were translated slightly less than one-half as efficiently as species s4; species m3, l2, and l3 one-quarter to one-third as efficiently; and species s1 one-twentieth to one-tenth as efficiently. Finally, species l1 and m1 were translated very inefficiently under all conditions; their translation efficiencies were usually no more than 1% of that of species s4.

Animals↗

PubMed2004

Multidirectional kinematics of the glenohumeral joint during simulated simple translation tests: impact on clinical diagnoses.

At the end ranges of motion, the glenohumeral capsule limits translation of the humeral head in multiple directions. Since the 6-degree of freedom kinematics of clinical tests are commonly utilized to diagnose shoulder injuries, the objective of this study was to determine the magnitude and repeatability of glenohumeral joint kinematics during a simulated simple anteroposterior translation test in the anterior and posterior directions. A magnetic tracking system was used to determine the kinematics of the humerus with respect to the scapula in eight cadaveric shoulders. At 60 degrees of glenohumeral abduction and 0 degrees of flexion/extension, a clinician applied anterior and posterior loads to the humerus at 0 degrees, 30 degrees, and 60 degrees of external rotation until a manual maximum (simulating a simple translation test) was achieved. Prior to each test, the reference position of the humerus shifted posteriorly 1.8+/-2.0 and 4.1+/-3.8 mm at 30 degrees and 60 degrees of external rotation, respectively. Anterior translation decreased significantly (p < 0.05) from 18.2+/-5.3 mm at 0 degrees of external rotation to 15.5+/-5.1 and 9.9+/-5.5 mm at 30 degrees and 60 degrees, respectively. However, no significant differences were detected between the posterior translations of 13.4+/-6.4, 17.1+/-5.0, and 15.8+/-6.0 mm at 0 degrees, 30 degrees, and 60 degrees of external rotation, respectively. Coupled translations (perpendicular to the direction of loading) at 0 degrees (6.1+/-4.0 and 3.8+/-2.9 mm), 30 degrees (4.7+/-2.7 and 5.9+/-3.1 mm), and 60 degrees (2.3+/-2.3 and 5.0+/-3.5 mm) of external rotation were in the inferior direction in both the anterior and posterior directions, respectively. Based on the data obtained, performing a simulated simple translation test should result in coupled inferior translations and anterior translations that are a function of external rotation. The low standard deviations demonstrate that the observed translations should be repeatable. Furthermore, capsular stretching or injury to the anterior-inferior region of the capsule should be detectable during clinical examination if excessive coupled translations exist or no posterior shift of the reference position with external rotation is noted.

Biomechanical Phenomena↗

PubMed2004

The role of baculovirus apoptotic suppressors in AcMNPV-mediated translation arrest in Ld652Y cells.

Infecting the insect cell line IPLB-Ld652Y with the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, which correlates with the presence of the AcMNPV apoptotic suppressor, p35. In this study, we investigated the role of apoptotic suppression on AcMNPV-induced translation arrest. Infecting cells with AcMNPV bearing nonfunctional mutant p35 did not result in global translation arrest. In contrast, global translation arrest was observed in cells infected with AcMNPV in which p35 was replaced with Opiap, Cpiap, or p49, baculovirus apoptotic suppressors that block apoptosis by different mechanisms than p35. These results indicated that suppressing apoptosis triggered translation arrest in AcMNPV-infected Ld652Y cells. Experiments using the DNA synthesis inhibitor aphidicolin and temperature shift experiments, using the AcMNPV replication mutants ts8 and ts8deltap35, indicated that translation arrest initiated during the early phase of infection, but events during the late phase were required for global translation arrest. Peptide caspase inhibitors could not substitute for baculovirus apoptotic suppressors to induce translation arrest in Ld652Y cells infected with a p35-null virus. However, if the p35-null-AcMNPV also carried hrf-1, a novel baculovirus host range gene, progeny virus was produced and treatment with peptide caspase inhibitors enhanced translation of a late viral gene transcript. Together, these results indicate that translation arrest in AcMNPV-infected Ld652Y cells is due to the anti-apoptotic function of p35, but suggests that rather than simply preventing caspase activation, its activity enhances signaling to a separate translation arrest pathway, possibly by stimulating the late stages of the baculovirus infection cycle.

Amino Acid Sequence↗

PubMed1998

An in vivo study of voluntary mandibular lateral translation concerning its existence, magnitude, and timing.

STATEMENT OF PROBLEM: The existence of mandibular lateral translation and the approaches to its measurement and interpretation by using a pantograph are controversial. PURPOSE: This study evaluated the validity of using a pantograph to measure mandibular lateral translation and analyzed human pantographic tracings to determine whether they exhibited mandibular lateral translation. MATERIAL AND METHODS: A pantograph was modified by adding 2 posterior horizontal recording tables and styli at the transverse horizontal axis. Pantographic tracings of 25 human subjects were compared with the corresponding theoretically determined values for tracings that exhibited only rotation with no translation. Differences in the tracings at 2 pantographic recording table locations, relative to the transverse horizontal axis, were also compared. RESULTS: The character of the lateral component of 100 pantographic tracings all differed from the lateral component of theoretically determined values for pure rotation. In 64% of tracings, over 50% of the total mandibular lateral translation occurred by the first 1 mm of forward movement of the nonworking side condyle. In 94% of tracings, more than 50% of the translation had occurred in the first 3 mm of forward movement. For the pantographic system used, the amount of mandibular translation represented in the tracing was not changed by altering the posterior horizontal recording table position in the anterior-posterior direction, relative to the transverse horizontal axis. CONCLUSION: All subjects showed evidence of mandibular lateral translation. New definitions for timing of mandibular lateral translation are proposed. Of the tracings, 64% were classified as exhibiting early translation, 30% as intermediate, and 4% as late mandibular lateral translation.

Adult↗

PubMed1999

Lumbar coupling during lateral translations of the thoracic cage relative to a fixed pelvis.

OBJECTIVE: To determine lumbar coupling during lateral postural translations (lumbosacral list) of the thoracic cage relative to a fixed pelvis. DESIGN: Digitized measurements from anteroposterior lumbar radiographs of 17 volunteers were obtained in neutral, maximal left lateral translation and maximal right lateral translation posture of the thoracic cage compared to a fixed pelvis. Subjects were constrained with two sets of clamps at the lateral borders of the pelvis and lower ribs. BACKGROUND: Data. Clinically, lumbosacral list is a common posture. Range of motion and spinal coupling results have not been reported for the lumbosacral list movement. METHODS: Four vertebral body corners, mid narrow-waisted body margins, superior and inferior pedicle margins, and spinous-lamina junction of T12-L5 were digitized on 51 anterior-posterior lumbar radiographs. Using the orthogonal axes of positive x-direction to the left, vertical as positive y, and anterior as positive z, digitized points were used to measure projected segmental z-axis rotation, y-axis rotation, and segmental lateral translations of each vertebra. RESULTS: Using the displacement of T12, subjects could translate 35-70 mm left or right along the x-axis with an average of 53.2 mm to the left and 52.1 mm to the right. Using superior endplates to superior sacral base, lateral flexion was largest at L1 and decreased from L1 to L5, but the segmental rotation angles for lateral flexion were largest at L2-L3 (3.9 degrees ), L3-L4 (6.2 degrees ) and L4-L5 (5.7 degrees ) and were in the same direction as the main motion translation. The relative z-axis rotation of T12 was opposite to the direction of L1-L5. The coupled y-axis rotations were less than 1 degrees and coupled segmental lateral translations were averaging less than 1 mm. CONCLUSIONS: Thoracic cage x-axis translations compared to a fixed pelvis are significant, between 35 and 70 mm. The z-axis lumbar coupled rotation was largest at L2-L3, L3-L4 and L4-L5 and to the same side of the main motion translation in L1-L5, but opposite the main motion direction for T12. All other movements were small, averaging less than 1 degrees or 1 mm. RELEVANCE: The clinically common posture of lateral translation of the thoracic cage (lumbosacral list) is often associated with disc herniation. Yet normal lumbar coupling patterns and total range of motion of this movement have not been established in the literature. Normal values for lumbar segmental coupling on anterior-posterior lumbo-pelvic radiographs during trunk list might be important for an analysis of segmental instability since segmental translations were determined to be 1 mm or less.

Adult↗

PubMedDate not supplied

Translation of the glenohumeral joint with the patient under anesthesia.

This study was undertaken to determine glenohumeral translation in the anesthetized patient. Radiographically assessed humeral translation was measured in 18 members of a control group, 10 patients with anterior instability, and 10 patients with multidirectional instability. In the control group stress radiographs revealed anterior translation of 17% (percentage of the diameter of the glenoid from anterior to posterior), posterior of 26%, and inferior to 29% (percentage of diameter of glenoid from superior to inferior). In patients with anterior instability anterior translation was 29%, posterior translation was 21%, and inferior translation was 49%. In patients with multidirectional instability anterior translation was 28%, posterior translation was 52%, and inferior translation was 46%. Overlap in translation was seen between normal members and those with instability. The most practical method to grade anterior and posterior translation of the humeral head within the glenoid fossa involves a clinical assessment of where the center of the humeral head rests in reference to the glenoid rim.

Adolescent↗

PubMed1995

The translational repression mediated by the platelet-derived growth factor 2/c-sis mRNA leader is relieved during megakaryocytic differentiation.

Expression of the platelet-derived growth factor 2/c-sis gene is highly restricted and controlled at multiple levels. Its structured mRNA leader, which is unusually long (1022 nucleotides), serves as a potent translational inhibitor. One of the sites of PDGF2 synthesis is megakaryocytes, implying that PDGF2 translation efficiency is modulated during megakaryocytic differentiation. To study the role of the mRNA leader as a translational cis-modulator, the hybrid T7/vaccinia cytoplasmic expression system was used to disconnect between determinants controlling transcription, alternative splicing, and mRNA stability from those controlling translation. Chimeric transcripts in which the human PDGF2/c-sis mRNA leader positioned in frame upstream of a reporter gene were used to determine whether the mRNA leader can confer variable translational efficiencies during differentiation. It is demonstrated that there is a time window during megakaryocytic differentiation of K562 cells in which the strong translational inhibition by PDGF2/c-sis mRNA leader is relieved. The time course of the translational repression relief is similar to that of PDGF2/c-sis transcriptional induction during the differentiation process. A 179-nucleotides CG-rich fragment immediately upstream of the initiator AUG codon is necessary for coffering stringent modulation of the translational efficiency. In NIH3T3 overexpressing translation initiation factor eIF4E, the inhibitory effect of the mRNA leader of c-sis is not relieved, suggesting that the changes in the translational machinery during megakaryocytic differentiation are beyond eIF4E activity. The possible involvement of a 5'-end-independent translational mechanism is discussed.

Base Sequence↗

PubMed1997

Translational regulation of lipoprotein lipase by epinephrine involves a trans-acting binding protein interacting with the 3' untranslated region.

To better characterize the translational regulation of lipoprotein lipase (LPL) by epinephrine, cytoplasmic extracts were prepared from 3T3-L1 adipocytes, 3T3-F442A adipocytes, and other nonadipocyte cell lines (C2 cells, 3T3 fibroblasts, and Chinese hamster ovary cells). After treatment with epinephrine, cell extracts from the adipocytes inhibited LPL translation in an in vitro translation assay, whereas extracts from the C2 cells and 3T3 fibroblasts did not affect LPL translation. To identify the region on the LPL mRNA that controlled translation, in vitro translation was carried out using constructs containing different LPL sequences. Specific deletion of the first 50 (1601-1650) nucleotides of the 3' untranslated region (UTR) resulted in a loss of translation inhibition. The addition of LPL 3' UTR to a heterologous reporter gene construct resulted in an inhibition of translation. Inhibition of the reporter LPL 3' UTR translation was demonstrated by the addition of epinephrine-treated cell extracts to an in vitro translation assay, as well as by transfection of this construct into 3T3-F442A cells, followed by treatment of the cells with epinephrine. Competition for a trans-acting binding protein was demonstrated by the addition of sense mRNA strands corresponding to the proximal 135 nucleotides of the 3' UTR of LPL. To identify a RNA-binding protein, adipocyte extracts were incubated with 32P-labeled RNA sequences followed by RNase treatment. The epinephrine-treated cell extract protected a fragment of RNA when the RNA included sequences on the proximal 3' UTR of LPL. Cross-linking of this protected fragment and analysis by SDS-polyacrylamide gel electrophoresis revealed a protein that migrated at about 30 kDa. Thus, the addition of epinephrine to 3T3 adipocytes results in an inhibition of translation through the production of a RNA-binding protein that binds to a region on the proximal 3' UTR of the LPL mRNA.

3T3 Cells↗

PubMed2002

5' stem-loop of collagen alpha 1(I) mRNA inhibits translation in vitro but is required for triple helical collagen synthesis in vivo.

The 5' stem-loop is a conserved sequence element found around the translation initiation site of three collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III). We show here that the 5' stem-loop of collagen alpha1(I) mRNA is inhibitory to translation in vitro. The sequence 5' to the translation initiation codon, as a part of the 5' stem-loop, is also not efficient in initiating translation under competitive conditions. This suggests that collagen alpha1(I) mRNA may not be a good substrate for translation. Since the 5' stem-loop binds protein factors in collagen-producing cells, this binding may regulate its translation in vivo. We studied in vivo translation of collagen alpha1(I) mRNA after transfecting collagen alpha1(I) genes with and without the 5' stem-loop into Mov 13 fibroblasts. The mRNA with the alpha1(I) 5' stem-loop was translated into pepsin-resistant collagen, which was secreted into the cellular medium. This mRNA also produced more disulfide-bonded high molecular weight collagen found intracellularly. The mRNA in which the 5' stem-loop was mutated, but without affecting the coding region of the gene, was translated into pepsin-sensitive collagen and produced only trace amounts of disulfide-bonded collagen. This suggests that the 5' stem-loop is required for proper folding or stabilization of the collagen triple helix. To our knowledge this is the first example that an RNA element located in the 5'-untranslated region is involved in synthesis of a secreted multisubunit protein. We suggest that 5' stem-loop, with its cognate binding proteins, targets collagen mRNAs for coordinate translation and couples translation apparatus to the rest of the collagen biosynthetic pathway.

5' Untranslated Regions↗

PubMed1992

Variable inhibition of cell-free translation by HIV-1 transcript leader sequences.

The 5' ends of all human immunodeficiency virus type I (HIV-1) transcripts have the potential to coordinately regulate translation of HIV-1 mRNAs. Conflicting observations of the translational impact of these sequences in various systems stimulated these analyses of translation in reticulocyte lysates. We report a sensitive, rapid, quantitative, and inexpensive cell-free translation assay in which translational efficiency is monitored by enzymatic assay of the translation products. Using this assay and conventional radiolabeling assays, we demonstrate that the HIV-1 transcript leader inhibits downstream translation and that the stem-loop structure is required. Under our assay conditions, this inhibition occurs predominantly in cis and is not mediated by the 68 kD, interferon-induced, double-stranded RNA-activated kinase (p68). However, under other assay conditions the HIV-1 leader may activate p68 and inhibit translation in trans. We show that variation between individual preparations of cell-free extracts can dramatically alter the magnitude of the translational inhibition by the HIV-1 leader. Further, we provide evidence that a heat-labile factor is required for efficient translation of transcripts containing the HIV-1 leader. These observations provide a foundation for identifying factors required for translation of HIV-1 transcripts.

Cell-Free System↗

PubMed1997

Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.

Animals↗

PubMed2006

Translating instruments for cross-cultural studies in headache research.

OBJECTIVE: To describe translation methods, to outline current recommendations for translation and to give an overview of translation procedures used in headache. BACKGROUND: In health-care research, particularly in the domain of health-related quality of life, an increasing number of questionnaires are translated for cross- cultural comparison. A number of headache-related questionnaires have also been translated. To ensure true cross-cultural comparison, it is important to use rigorous translation methods to ensure a high quality translated version of the instruments. METHODS: Literature relevant to cross-cultural translations was reviewed, as well as literature concerning translation of headache-related instruments. CONCLUSION: More information on the translation process for headache-related instruments is essential to carry out high quality cross-cultural research. Recommendation are given to encourage good translation practices.

Cross-Cultural Comparison↗

PubMed2006

trans regulation of cap-independent translation by a viral subgenomic RNA.

Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.

3' Untranslated Regions↗

PubMed1978

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.

Aurintricarboxylic Acid↗

PubMed2000

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA.

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

5' Untranslated Regions↗