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PubMed1994

Post-transcriptional regulation of the str operon in Escherichia coli. Structural and mutational analysis of the target site for translational repressor S7.

In the Escherichia coli str operon, translation of the S12 and S7 genes is largely coupled, and the translational repressor S7 inhibits S7 translation, which is coupled to that of S12, but does not inhibit independent translation of S7 by free ribosomes in the intracellular pool. We have studied the S12-S7 intercistronic region of mRNA by analyzing RNA synthesized in vitro using structure-specific nucleases and a chemical probe, dimethyl sulfate. Based on the results obtained, we have deduced a secondary structure model of the S12-S7 intercistronic region and identified nucleotide residues "protected" by S7. We then carried out site-directed mutagenesis to identify nucleotide residues important for S7 translation as well as for repression by S7. The results showed that two distinct regions are important for S7-mediated repression; one is the S7 binding region identified by the protection analysis and the second is the stem structure that sequesters the Shine-Dalgarno sequence for the S7 gene. Some of the base alterations in the first region abolished S7 binding and, as a consequence, abolished S7-mediated repression, without affecting the efficiency of S7 translation. Other mutations disrupting the stem structure in the second region abolished S7-mediated repression without significantly affecting the S7-mRNA interaction. We also found that certain mutations drastically decrease S7 translation achieved by translational coupling without affecting S7 translation achieved by independent initiation. These mutations are in base-paired regions and evidence was obtained to suggest that these base-paired structures are important for translational coupling. We suggest that some specific RNA structures in the intercistronic region play an active role in achieving translational coupling in this system, and that repression of S7 translation by S7 protein is due to disruption of such structures induced by binding of S7 protein to the target site, rendering translational coupling very inefficient, but leaving independent translation initiation unaffected.

Base Sequence↗

PubMed2006

Preferential translation of vesicular stomatitis virus mRNAs is conferred by transcription from the viral genome.

Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

Genome, Viral↗

PubMed1994

Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1999

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro.

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.

3C Viral Proteases↗

PubMed2001

The reovirus S4 gene 3' nontranslated region contains a translational operator sequence.

Reovirus mRNAs are efficiently translated within host cells despite the absence of 3' polyadenylated tails. The 3' nontranslated regions (3'NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3'NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3'NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product sigma3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3'NTR resulted in a 20 to 50% decrease in sigma3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3'NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3'NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3'NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3'NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3'NTR. These results indicate that the reovirus S4 gene 3'NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3'NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs.

3' Untranslated Regions↗

PubMedDate not supplied

Anterior tibial translation during a maximum quadriceps contraction: is it clinically significant?

Quadriceps exercises are used sparingly in the early rehabilitation of ACL reconstructions because of concern about prematurely stretching the ACL graft. The aim of this study was to determine if a maximum isometric quadriceps contraction significantly translates the tibia anteriorly at 15 degrees, 30 degrees, 45 degrees, 60 degrees, and 75 degrees of flexion. Secondly, the role of the ACL in knee stability was analyzed by comparing the amount of tibial translation in normal, ACL deficient, and reconstructed knees. Thirdly, the location in the motion arc where a quadriceps contraction produces anterior tibial translation was determined. Anterior tibial translation was measured using an arthrometer (KT-1000) during an 89 N and manual maximum translation applied to the knee at rest. The manual maximum translation test determines the magnitude of anterior tibial translation produced by a high anterior force applied directly to the proximal calf. These translations were compared to the tibial translation intrinsically induced by a quadriceps contraction. Testing was performed in normal (N = 22), ACL deficient (N = 10), and reconstructed (N = 10) knees. Anterior tibial translation produced by a maximum quadriceps contraction was measured at 15 degrees, 30 degrees, 45 degrees, 60 degrees, and 75 degrees of flexion. The extension exercise resulted in less anterior tibial displacement than an 89 N drawer and half the translation produced by a manual maximum translation (P less than 0.001). Instrumented laxity testing produced greater anterior translation of the tibia than a maximum isometric quadriceps contraction. Anterior tibial translation was the same during maximum isometric knee extension in all tested knees.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2004

Instrument translation process: a methods review.

BACKGROUND: Cross-cultural and international collaborative studies are needed in nursing research. Therefore, it is necessary to translate research instruments into the language of the culture being studied. In this methods review, different processes of instrument translation and evaluation of translation adequacy in published nursing research are described and classified into a hierarchy. METHODS: Studies including translation of quantitative research instruments were reviewed. Forty-seven studies were included. These were classified into six categories. RESULTS: Studies were classified into categories as follows: forward-only translation (2), forward-only translation with testing (7), back-translation (13), back-translation with monolingual test (18), back-translation with bilingual test (3), and back-translation with both monolingual and bilingual test (4). Strengths and weaknesses are analysed. CONCLUSION: The studies reviewed used diverse methods of varying quality. There is need for consensus among researchers in how to achieve quality of instrument translation in cross-cultural research. Researchers should carefully attend to achieving and reporting evidence of the accuracy and validity of instrument translation. When back-translation fails to achieve semantic equivalence, the instrument development process should be replicated in the target language.

Cross-Cultural Comparison↗

PubMed2000

A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation.

Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5' untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

5' Untranslated Regions↗

PubMed1995

Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding.

The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.

Bacterial Proteins↗

PubMed1984

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system.

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-to-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.

Acetyltransferases↗

PubMed2006

In vitro selection of translational regulatory elements.

Untranslated regions (UTRs) of mRNAs carry various kinds of translational regulatory elements; however, our knowledge of them is still limited. We created an in vitro selection system that allows us to make a systematic enrichment of the sequences that alter translation efficiency (SESTRE) in any given mRNA and translation system. This method consists of the introduction of random nucleotide sequences into the UTRs of given mRNAs, followed by translation, size fractionation of the polyribosomes, and reverse transcription and PCR amplification (RT-PCR), with repeated cycles of these steps to enrich highly or poorly translatable mRNAs. With this experimental method, we examined how and where translational enhancer motifs emerge on mRNAs using the in vitro translation systems of wheat germ extract. The results indicate that the translational enhancers differentially emerge in response to the presence or absence of the 5' cap. Interestingly, the translational enhancers that activate cap-independent translation evolved more readily in the 3' UTR than in the 5' UTR in wheat germ extract. This SESTRE method should be a powerful tool with which to improve the translational efficiency of given mRNAs in given translation systems and to investigate the structure-function relationship of eukaryotic mRNAs underlying translational control.

3' Untranslated Regions↗

PubMedDate not supplied

Intraoperative measurement of shoulder translation.

Assessing laxity of the shoulder joint in patients who are under anesthesia is a standard procedure before arthroscopy. The aim of this study was to evaluate a novel instrument for quick and reliable intraoperative measurement of glenohumeral translation. Previous testing of various designs has resulted in a device secured by 1 pin in the acromion and 1 pin in the proximal humerus. These pins are interconnected by a sliding ruler that gives translation values in millimeter increments as the laxity tests are performed. Comparison between manual arbitrary approximation of laxity and instrumented translation measurements showed that manual testing is reasonably good for assessment of anterior and posterior translation, without, however, providing values of translation in millimeter increments. The low correlation between manual assessment and instrumented inferior translation measurements indicates that inferior translation is more difficult to approximate manually. The shoulder translation tester was used in 102 patients. The mean values for clinically stable shoulders (n = 58) were 5 mm for anterior translation, 5 mm for posterior translation, and 4 mm for inferior translation. The corresponding values in unstable shoulders were significantly higher than in the stable shoulders, especially in patients with multidirectional instability. We conclude that the shoulder translation tester is easy and quick to use. It provides quantitative values of translation and will thus contribute information for correct diagnosis, therapy, and documentation.

Adult↗

PubMed1995

A sequence located 4.5 to 5 kilobases from the 5' end of the barley yellow dwarf virus (PAV) genome strongly stimulates translation of uncapped mRNA.

An infectious, in vitro transcript from a full-length cDNA clone of the barley yellow dwarf virus (PAV serotype) genome translated efficiently in a wheat germ translation extract. Deletions in a region that we call the 3' translational enhancer, located between bases 4,513 and 5,009 in the 5,677-base genome, reduced translation of the 5'-proximal open reading frames from uncapped RNA by at least 30-fold. Deletions elsewhere in all but the 5' end of the genome had no effect on translation. Presence of a m7G(5')pp(5')G cap on the 5' end fully restored translational efficiency of transcripts lacking the 3' translational enhancer. The translation enhancer reduced inhibition of translation by free cap analog, did not affect RNA stability, and did not function in reticulocyte lysates. When placed in the 3'-untranslated region of uncapped mRNA encoding the beta-glucuronidase gene, the translation enhancer stimulated translation more than 80-fold, in the presence of the viral, but not a plasmid-derived, 5' leader. A polyadenylate tail could not substitute for the 3' translation enhancer. These observations provide an extreme example, in terms of distance from the 5' end and level of stimulation, of an mRNA in which a sequence near the 3' end stimulates translation.

Enhancer Elements, Genetic↗

PubMed2003

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts.

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.

Chloroplasts↗

PubMed1989

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation.

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Animals↗

PubMed2004

Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting.

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.

DNA-Directed RNA Polymerases↗

PubMed1995

Regulation of lipoprotein lipase translation by epinephrine in 3T3-L1 cells. Importance of the 3' untranslated region.

Lipoprotein lipase (LPL) is a central enzyme in lipoprotein metabolism and is in part responsible for adipocyte lipid accumulation. Catecholamines are known to decrease the activity of LPL in adipocytes, and we have previously demonstrated that this inhibition occurs posttranscriptionally, with a prominent inhibition of LPL translation. To better characterize the inhibition of LPL translation, 3T3-L1 cells were differentiated into adipocytes, and exposed to epinephrine. Epinephrine induced a dose-dependent decrease in LPL synthesis using [35S]methionine incorporation, with no change in LPL mRNA levels, demonstrating translational regulation of LPL in this cell line. The poly A-enriched RNA from epinephrine-treated cells was translated well in vitro, and there was no difference in the polysome profiles from control and epinephrine-treated cells, suggesting that epinephrine did not affect mRNA editing, and did not induce an inhibition of translation initiation. To obtain evidence for the presence of an inhibitory factor, a cytoplasmic extract from control, and epinephrine-treated adipocytes was human. When compared to the control cell extract, the epinephrine-treated cell extract sharply inhibited LPL translation in vitro, yet had no effect on the translation of other mRNAs. Epinephrine-treated cells had fourfold more of this inhibitor activity than control cells, and this translation inhibition was partially reversed by heat treatment. To determine what region of the LPL mRNA was involved in the translation inhibition, different LPL constructs were synthesized. The inhibitory effect of the epinephrine-treated cell extract was dependent on the presence of the first 40 nucleotides of the 3' (untranslated region UTR) (nucleotides 1599-1638), whereas deletion of the 5' UTR and other areas of the 3' UTR had no effect on translation inhibition. When a sense RNA strand corresponding to this region was added to the in vitro translation reaction, it restored translation towards normal, suggesting that the sense strand was competing for a transacting binding protein. Thus, epinephrine-treated adipocytes produced a transacting factor, probably a protein, that interacted with a region on the LPL mRNA between nucleotides 1599 and 1638, resulting in an inhibition of translation. These studies add new insight into the hormonal regulation of LPL.

3T3 Cells↗

PubMed1997

Glenohumeral joint translations before and after total shoulder arthroplasty. A study in cadavera.

UNLABELLED: The purpose of the present study was to examine the motions of natural and prosthetically reconstructed glenohumeral joints without capsular contracture in the laboratory to obtain a better understanding of joint motion as it may relate to failure of the implant. Seven joints from fresh-frozen human cadavera were instrumented with a six-degrees-of-freedom magnetic tracking device to study patterns of translation and rotation with and without the application of simulated muscle forces (active and passive models). The specimens were tested before and after reconstruction of the joint with use of modified operative techniques and implants that had from zero to five millimeters of radial mismatch between the humeral head and glenoid components. The natural and reconstructed joints had similar patterns of translation, with larger rotations and translations observed in the passive model. On the average, the active translations of the natural joints were best reproduced by the reconstructed joints with less conforming articulations. The mean active translation in the natural joints was 1.5 millimeters along the anterior-posterior axis and 1.1 millimeters along the superior-inferior axis. The active translations in the reconstructed joints were observed to increase consistently as the conformity of the components decreased: the mean active anterior-posterior translations ranged from 0.3 millimeter for conforming components to 1.7 millimeters for components with a five-millimeter radial mismatch, and the mean active superior-inferior translations ranged from 0.4 to 1.1 millimeters. The patterns of translation during passive motions were less consistent, presumably because of the influence of capsular ligaments and because the translations were large enough for the articular surface of the humeral head to lose contact with the articular surface of the glenoid component. CLINICAL RELEVANCE: Glenohumeral translations during active motions were found to depend on articular conformity, which indicates that the choice of implants may have important consequences for normal motions. Totally conforming designs may impose a degree of restraint to translations that is higher than that in the natural joint. The patterns of translation may influence the longevity of the joint replacement with respect to stability, loosening of the glenoid component, and wear of the components.

Aged↗