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PubMed2000

Genetic organization of the downstream region of the mecA element in methicillin-resistant Staphylococcus aureus isolates carrying different polymorphisms of this region.

We describe here the genetic organization of the mec element downstream of the mecA gene in 34 different methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates carrying 13 of the most frequent polymorphisms of mecA and representing the major epidemic clones of MRSA. All polymorphisms carried three common genetic elements: the hypervariable region, a copy of IS431, and a unique 2-kb sequence (downstream constant segment, or dcs) for which no homologous sequences are found in data banks. Polymorphisms of the downstream region were shown to be caused by the presence of linearized plasmids flanked by insertion sequences (pUB110, pT181, and pI258) and the autonomous insertion sequence IS256.

Bacterial Proteins↗

PubMed1994

Integration host factor and sequences downstream of the Pseudomonas aeruginosa algD transcription start site are required for expression.

Pseudomonas aeruginosa is an extremely important opportunistic pathogen in immunocompromised individuals. Strains of P. aeruginosa isolated from chronic lung infections in patients with the genetic disease cystic fibrosis have a mucoid colony morphology. This phenotype is due to overproduction of the exopolysaccharide alginate, which is believed to confer a selective advantage on P. aeruginosa in cystic fibrosis lungs. Alginate biosynthesis is controlled by a complex regulatory mechanism. Genes located in the 34-min region of the P. aeruginosa chromosome form an operon which encodes most of the biosynthetic enzymes necessary for alginate production. algD, the first gene in the operon and a critical point for the transcriptional regulation of alginate biosynthesis, is controlled by several trans, cis, and environmental factors. In this study, the involvement of the histone-like protein integration host factor (IHF) in algD expression was examined. Sequences with similarity to consensus IHF-binding sites of Escherichia coli were identified 75 bp upstream (site 1) and 90 bp downstream (site 2) of the start of algD transcription. In gel band mobility shift assays, DNA fragments containing either site bind IHF but site 2 has an approximately 90-fold higher affinity for IHF. Mutations in each of the elements were generated, and they resulted in the reduction or loss of in vitro IHF binding and a three- to fourfold decrease in algD-cat expression. This indicates that IHF binding is necessary for high-level algD transcription. The presence of a high-affinity IHF-binding site located 3' of the algD transcription start site suggested that sequences further downstream of this element are involved in algD expression. When a fragment located downstream of site 2 and upstream of the promoterless cat gene (+110 to +835) was deleted, algD-cat expression was reduced 10-fold supporting the notion that 3' enhancer elements are required for algD transcription. This is the first direct evidence of a 3' element involved in the control of a P. aeruginosa gene. It is postulated that IHF mediates the formation of a higher-order looped structure which is necessary for efficient algD transcription.

Bacterial Proteins↗

PubMed1996

Long-distance effect of downstream transcription on activity of the supercoiling-sensitive leu-500 promoter in a topA mutant of Salmonella typhimurium.

Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.

DNA Topoisomerases, Type I↗

PubMed1996

The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful.

Amino Acid Sequence↗

PubMed1991

Enhancement of RNA polymerase II initiation complexes by a novel DNA control domain downstream from the cap site of the cytomegalovirus major immediate-early promoter.

The major immediate-early promoter (MIEP) of human cytomegalovirus is a remarkably strong RNA polymerase II transcription control unit. We have identified and characterized a novel regulatory domain associated with MIEP downstream from the initiation site of transcription. The downstream regulatory region was first identified by analyzing a series of mutations in the 5' untranslated leader exon. This regulatory domain was shown to enhance the number of functional initiation complexes without significantly altering the apparent elongation rate by RNA polymerase II transcription. In addition, run-off in vitro transcription and DNA-binding experiments identified two distinct downstream elements that specify the interaction of cellular transcription factors. One of these elements contains a reiterated sequence motif, present twice within the leader exon. The second element is an 18-bp sequence located at approximately nucleotide position +33 that is conserved between strains of cytomegalovirus from different species. On the basis of two criteria, an oligonucleotide competition assay and oligomerization upstream of the promoter, the binding of factors to the conserved box was shown to be critical for mediating the level of transcription from MIEP. Two discrete cellular nuclear proteins, designated LTF A and B (for leader transcription factor A and B binding factors), were found to specifically recognize the conserved element. This study of promoter-proximal elements within transcribed sequences demonstrates the recognition of the control domain at the DNA level that functions to increase the number of committed RNA polymerase II transcription complexes.

Animals↗

PubMed1994

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.

Base Sequence↗

PubMed1995

Efficient excision of the upstream large intron from P4-generated pre-mRNA of the parvovirus minute virus of mice requires at least one donor and the 3' splice site of the small downstream intron.

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q. Zhao, R. V. Schoborg, and D. J. Pintel, J. Virol. 68:2849-2859, 1994). In this report, we show that the requirement of downstream small intron sequences is complex and that efficient excision of the upstream intron requires at least one small intron donor and the 3' splice site. In the absence of both small intron donors, a new spliced product is produced in which the intervening exon is skipped and the large intron donor at nucleotide 514 is joined to a small intron acceptor. Exon skipping caused by the loss of the two small intron donors can be overcome, and the excision of the large intron can be regained by mutations that improve the large intron polypyrimidine tract. These results are consistent with a model in which the binding of multiple splicing factors that assemble at both a downstream donor and acceptor facilitates the binding of splicing factors to the weak polypyrimidine tract of the upstream large intron, thereby defining the intervening exon and promoting excision of the upstream intron.

Animals↗

PubMed1996

Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.

Amino Acid Sequence↗

PubMed1997

Identification of sequences downstream of the primer binding site that are important for efficient replication of human immunodeficiency virus type 1.

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.

Binding Sites↗

PubMed1997

Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity.

When transcriptionally active, the human immunodeficiency virus (HIV) promoter contains a nucleosome-free region encompassing both the promoter/enhancer region and a large region (255 nucleotides [nt]) downstream of the transcription start site. We have previously identified new binding sites for transcription factors downstream of the transcription start site (nt 465 to 720): three AP-1 sites (I, II, and III), an AP3-like motif (AP3-L), a downstream binding factor (DBF) site, and juxtaposed Sp1 sites. Here, we show that the DBF site is an interferon-responsive factor (IRF) binding site and that the AP3-L motif binds the T-cell-specific factor NF-AT. Mutations that abolish the binding of each factor to its cognate site are introduced in an infectious HIV-1 molecular clone to study their effect on HIV-1 transcription and replication. Individual mutation of the DBF or AP3-L site as well as the double mutation AP-1(III)/AP3-L did not affect HIV-1 replication compared to that of the wild-type virus. In contrast, proviruses carrying mutations in the Sp1 sites were totally defective in terms of replication. Virus production occurred with slightly delayed kinetics for viruses containing combined mutations in the AP-1(III), AP3-L, and DBF sites and in the AP3-L and DBF-sites, whereas viruses mutated in the AP-1(I,II,III) and AP3-L sites and in the AP-1(I,II,III), AP3-L, and DBF sites exhibited a severely defective replicative phenotype. No RNA-packaging defect could be measured for any of the mutant viruses as determined by quantification of their HIV genomic RNA. Measurement of the transcriptional activity of the HIV-1 promoter after transient transfection of the HIV-1 provirus DNA or of long terminal repeat-luciferase constructs showed a positive correlation between the transcriptional and the replication defects for most mutants.

Base Sequence↗

PubMed2000

Expression of the Totivirus Helminthosporium victoriae 190S virus RNA-dependent RNA polymerase from its downstream open reading frame in dicistronic constructs.

The undivided double-stranded RNA (dsRNA) genome of Helminthosporium victoriae 190S virus (Hv190SV) (genus Totivirus) consists of two large overlapping open reading frames (ORFs). The 5'-proximal ORF encodes a capsid protein (CP), and the downstream, 3'-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and Schizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

Capsid↗

PubMed2002

The oncogenic protein kinase Tpl-2/Cot contributes to Epstein-Barr virus-encoded latent infection membrane protein 1-induced NF-kappaB signaling downstream of TRAF2.

The Epstein-Barr virus-encoded latent infection membrane protein 1 (LMP1) is a pleiotropic protein, the activities of which include effects on cell transformation and phenotype, growth, and survival. The ability of LMP1 to mediate at least some of these phenomena could be attributed to the activation of the transcription factor NF-kappaB. LMP1 promotes NF-kappaB activation through the recruitment of the adapter protein TRAF2 and the formation of a dynamic multiprotein complex that includes the NF-kappaB kinase, the IkappaB kinases, and their downstream targets, IkappaBs and p105. In this study, we have identified the oncogenic kinase Tpl-2/Cot as a novel component of LMP1-induced NF-kappaB signaling. We show that Tpl-2 is expressed in primary biopsies from patients with nasopharyngeal carcinoma and Hodgkin's disease, where LMP1 is also found. Inducible expression of LMP1 promotes the activation of Tpl-2, and a catalytically inactive Tpl-2 mutant suppresses LMP1-induced NF-kappaB signaling. In colocalization and coimmunoprecipitation experiments, Tpl-2 and TRAF2 were found to interact with Tpl-2 functioning downstream of TRAF2. Consistent with this observation, catalytically inactive Tpl-2 also blocked CD40-mediated NF-kappaB activation, which largely depends on TRAF2. The ability of Tpl-2 to influence LMP1-induced NF-kappaB occurs through modulation of both IkappaBalpha and p105 functions. Furthermore, Tpl-2 was found to influence the expression of angiogenic mediators, such as COX-2 in LMP1-transfected cells. These data identify Tpl-2 as a component of LMP1 signaling downstream of TRAF2 and as a modulator of LMP1-mediated effects.

3T3 Cells↗

PubMed2006

Two G-rich regulatory elements located adjacent to and 440 nucleotides downstream of the core poly(A) site of the intronless melanocortin receptor 1 gene are critical for efficient 3' end processing.

Cleavage and polyadenylation is an essential processing reaction required for the maturation of pre-mRNAs into stable, export- and translation-competent mature mRNA molecules. This reaction requires the assembly of a multimeric protein complex onto a bipartite core sequence element consisting of an AAUAAA hexamer and a GU/U-rich downstream sequence element. In this study we have analyzed 3' end processing of the human melanocortin 1 receptor gene (MC1R). The MC1R gene is an intron-free transcription unit, and its poly(A) site lacks a defined U/GU-rich element. We describe two G-rich sequence elements that are critical for efficient cleavage at the MC1R poly(A) site. The first element is located 30 nucleotides downstream of the cleavage site and acts as an essential closely positioned enhancer. The second G-rich region is positioned more than 440 nucleotides downstream of the MC1R processing site and is instrumental for optimal processing efficiency. Both G-rich sequences contain clusters of heterogeneous nuclear ribonucleoprotein binding motifs and act together to enhance cleavage at the MC1R poly(A) site.

3' Untranslated Regions↗

PubMed1990

A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.

Base Sequence↗

PubMed1992

Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element.

The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.

Adenoviruses, Human↗

PubMed1993

Contribution of sequences downstream of the TATA element to a protein-DNA complex containing the TATA-binding protein.

A TATA complex that forms on the hsp70 promoter has been found to depend on sequence-specific interactions that occur at the transcription start and regions further downstream. The complex was detected with a gel shift assay and further characterized with interference assays. Antibodies reveal that the TATA-binding protein is in the complex. Interference assays localize specific contacts in the TATA element, the start site, and in a region approximately 25 bp downstream of the start site that contribute to either the assembly or the maintenance of the complex. Contact at the TATA element is made in the minor groove, as has been reported for the recombinant TATA-binding protein. Mutation in the TATA element or the start site of hsp70 causes complex formation to be more strongly dependent on contacts in the +25 region than in the normal core promoter. Examination of the hsp26 and histone H4 genes indicates that similar contacts contribute to the TATA complexes that form on these promoters. The results suggest that specific contacts downstream of the TATA element could play a key role in establishing the transcriptional potential of a gene by contributing to the interaction of the TATA-binding protein.

Animals↗

PubMed1994

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Animals↗

PubMed1994

Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron.

Translational control of the GCN4 gene in response to amino acid availability is mediated by four short open reading frames in the GCN4 mRNA leader (uORFs) and by phosphorylation of eukaryotic initiation factor 2 (eIF-2). We have proposed that reducing eIF-2 activity by phosphorylation of its alpha subunit or by a mutation in the eIF-2 recycling factor eIF-2B allows ribosomes which have translated the 5'-proximal uORF1 to bypass uORF2 to uORF4 and reinitiate at GCN4 instead. In this report, we present two lines of evidence that all ribosomes which synthesize GCN4 have previously translated uORF1, resumed scanning, and reinitiated at the GCN4 start site. First, GCN4 expression was abolished when uORF1 was elongated to make it overlap the beginning of the GCN4 coding region. Second, GCN4 expression was reduced as uORF1 was moved progressively closer to GCN4, decreasing to only 5% of the level seen in the absence of all uORFs when only 32 nucleotides separated uORF1 from GCN4. We additionally found that inserting small synthetic uORFs between uORF4 and GCN4 inhibited GCN4 expression under derepressing conditions, confirming the idea that reinitiation at GCN4 under conditions of diminished eIF-2 activity is proportional to the distance of the reinitiation site downstream from uORF1. While uORF4 and GCN4 appear to be equally effective at capturing ribosomes scanning downstream from the 5' cap of mRNA, these two ORFs differ greatly in their ability to capture reinitiating ribosomes scanning from uORF1. When the active form of eIF-2 is present at high levels, reinitiation appears to be much more efficient at uORF4 than at GCN4 when each is located very close to uORF1. Under conditions of reduced recycling of eIF-2, reinitiation at uORF4 is substantially suppressed, which allows ribosomes to reach the GCN4 start site; in contrast, reinitiation at GCN4 in constructs lacking uORF4 is unaffected by decreasing the level of eIF-2 activity. This last finding raises the possibility that time-dependent binding to ribosomes of a second factor besides the eIF-2-GTP-Met-tRNA(iMet) ternary complex is rate limiting for reinitiation at GCN4. Moreover, our results show that the efficiency of translational reinitiation can be strongly influenced by the nature of the downstream cistron as well as the intercistronic distance.

Cloning, Molecular↗