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1.
The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It 总被引:4,自引:2,他引:2
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Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control. 相似文献
2.
A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae 总被引:9,自引:0,他引:9
Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA. Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon. Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4. This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4. Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4. Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4. Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1. This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4. The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1. In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression. 相似文献
3.
Positions +5 and +6 can be major determinants of the efficiency of non-AUG initiation codons for protein synthesis. 总被引:11,自引:6,他引:11
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Only rarely do GUG (or CUG or ACG) codons which precede the 5'-proximal AUG function as initiators of protein synthesis, even when they are within a context that contains a purine at position -3 and a G at +4. For example, the upstream GUG of the human parainfluenza virus type 1 (hPIV1) P gene is initiated by ribosomes at high frequency, whereas a seemingly similar GUG codon in the hPIV3 P gene is not used at all. We have examined the reasons for this by expressing chimeric hPIV3/hPIV1 mRNAs, both in vivo and in vitro. A major determinant for efficient GUG utilization was located downstream of the GUG, but this did not appear to be involved in the formation of secondary structure. Rather, the sequence immediately downstream was found to be critical; this determinant was mapped to positions +5 and +6. GUG could be used efficiently for ribosomal initiation when the second codon was GAU but not when it was GUA. Similar results were found when other non-AUG start sites, the Sendai virus P gene ACG and the c-myc-1 CUG, were examined. These results suggest that positions +5 and +6 are important determinants for initiation at non-AUG start sites, and that they are recognized independently of the overall secondary structure of the mRNA. 相似文献
4.
Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus
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Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses. 相似文献
5.
6.
Mikhail Schepetilnikov Gregory Schott Konstantina Katsarou Odon Thiébeauld Mario Keller Lyubov A. Ryabova 《Nucleic acids research》2009,37(17):5838-5847
The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs. 相似文献
7.
8.
The post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non-permissive temperature of a temperature-sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3' end of mRNA. However, instead of accumulating at the 3' end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer-messenger RNA from the 3' end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis. 相似文献
9.
Sequences 5' of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation. 总被引:1,自引:0,他引:1
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Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell. Ribosomes which translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. Under non-starvation conditions reinitiation occurs at one of the remaining three uORFs and GCN4 is repressed. Under starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at GCN4 instead. The high frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1 also strongly enhance reinitiation. First, reinitiation was severely inhibited when uORF1 was transplanted into the position of uORF4, even though the native sequence environment of the uORF1 stop codon was maintained, and this effect could not be accounted for by the decreased uORF1-GCN4 spacing. Second, insertions and deletions in the leader preceding uORF1 greatly reduced reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of ribosome release following peptide termination at uORF1. Alternatively, they may facilitate rebinding of an initiation factor required for reinitiation prior to resumption of the scanning process. 相似文献
10.
Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli
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Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation. 相似文献
11.
Ryabova LA Pooggin MM Dominguez DI Hohn T 《The Journal of biological chemistry》2000,275(47):37278-37284
The pathways of scanning ribosome migration controlled by the cauliflower mosaic virus 35 S RNA leader were investigated in vitro and in vivo. This long (600 nucleotides) leader contains several short open reading frames (sORFs) and folds into an extended hairpin structure with three main stable stem sections. Translation initiation downstream of the leader is cap-dependent and occurs via ribosomal shunt under the control of two cis elements, a short open reading frame A (sORF A) followed by stem section 1. Here we show that a second similar configuration comprising sORF B followed by stem section 2 also allows shunting. The efficiency of the secondary shunt was greatly increased when stem section 1 was destabilized. In addition, we present evidence that a significant fraction of reinitiation-competent ribosomes that escape both shunt events migrate linearly via the structured central region but are intercepted by internal AUG start codons. Thus, expression downstream of the 35 S RNA leader is largely controlled by its multiple sORFs. 相似文献
12.
Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA 总被引:1,自引:1,他引:0
TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides. 相似文献
13.
14.
Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus. 总被引:10,自引:8,他引:2
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RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported. 相似文献
15.
Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa. 总被引:1,自引:0,他引:1
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The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon. 相似文献
16.
Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo. 总被引:6,自引:0,他引:6
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G J Belsham 《The EMBO journal》1992,11(3):1105-1110
The initiation of protein synthesis on foot-and-mouth disease virus RNA occurs at two sites separated by 84 nucleotides. Immediately upstream from the first of these sites is the internal ribosome entry site (IRES), which directs the translation of this RNA to be cap-independent. The utilization of these two initiation sites has been examined using artificial fusion genes in vivo under a variety of conditions. Additional in-frame AUG codons have been introduced between these two authentic start sites to determine the mechanism by which ribosomes recognize the second start site. The results indicate that following internal entry of ribosomes on the 5' side of the first initiation codon, many fail to initiate protein synthesis at this position and scan along the RNA to the second initiation site. In the presence or absence of the IRES both initiation sites are efficiently used but the utilization of the two sites is slightly biased towards the second initiation site by the IRES. Furthermore, in the presence of the IRES, protein synthesis initiates at both sites independently of the activity of the cap-binding complex. 相似文献
17.
It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts. 相似文献
18.
It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end. 相似文献
19.
Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals 总被引:10,自引:0,他引:10
D A Steege K C Cone C Queen M Rosenberg 《The Journal of biological chemistry》1987,262(36):17651-17658
20.
The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF. 相似文献