Functional analysis of lac repressor restart sites in translational initiation and reinitiation

To define some of the features that influence ribosomal recognition of translational restart sites in the lac repressor mRNA, recombinant DNA methods have been used to construct lacI-Z fusions in which lacZ gene expression is dependent upon initiation or reinitiation within lacI mRNA sequences. Reinitiation efficiencies, as assessed by beta-galactosidase levels in strains bearing such plasmids, appear to be determined by at least three features of the RNA between the termination codon and reinitiation codon: the presence of competing out-of-frame AUG or GUG triplets, the distance between termination and reinitiation points, and the extent to which restart sequences remain accessible to ribosomes. While some of the restart sites are used with substantial efficiency for reinitiation, they do not function detectably as primary initiators if placed at the 5' end of the lacZ mRNA. This finding concurs with our observation that relative to the wild-type initiator region, which is recovered in quantitative yield from in vitro initiation reactions, ribosome protection of the four restart sites occurs at more than 100-fold lower efficiencies. In part, the lack of initiation activity is rationalized by the striking potential these sequences have for forming stable secondary structures that sequester elements essential for ribosome binding. However, the differential functioning of the restart sites in primary initiation versus reinitiation must also reflect real differences in the mechanisms operative in the two events.     

Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence.

In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet. Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA. The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes. The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency. On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons. Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs.     

Mutants of translational components that alter reading frame by two steps forward or one step back

External suppressors, sufS, of a -1 frameshift mutant cause ribosomes to shift into the -1 frame when reading the sequence CAG GGA GUG. The resulting product is not Gln-Gly-Val but Gln-Gly-Ser with Ser being encoded by the underlined AGU. The alleles investigated are approximately 2% efficient in causing frameshifting. Two other suppressors, hopR and hopE of the same -1 frameshift mutant, cause some ribosomes reading the sequence GUG UG to decode a single amino acid, Val, from the five nucleotides. The possibility is considered that peptidyl-tRNA(Val) dissociates from the mRNA, but re-pairs in a triplet manner after the mRNA slips forward by two bases.     

Influences of bacterial DNA base-ratios and amino acid composition of the gene product on the consequences of frameshift mutations

Using as an example the E. coli lac I (repressor) gene product, it can be shown that DNA base-ratio is a major determinant of the detailed outcomes of both +1 (?2) and ?1 (+2) types of frameshift mutations. Potential reinitiation codons (AUG or GUG) and premature stop codons (UAA, UAG and UGA) occur in very different proportions depending on the type of frameshift and the DNA base-ratio.A comparison of the H. halobium bacteriorhodopsin precursor gene with other actual and theoretical genes reveals that the amino acid composition of the gene product is a second, important, determinant of the detailed outcome of frameshift mutations. Rules are formulated for the occurrence of particular codon, and hence amino acid, doublets in AT-GC-rich or intermediate base-ratio DNA as these affect frameshift-generated reinitiation and premature stop codons.     

Further characterisation of the translational termination-reinitiation signal of the influenza B virus segment 7 RNA

Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAAUG and ～10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAAUG, known as the 'termination upstream ribosome binding site' (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAAUG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.     

Truncated forms of the dual function human ASCT2 neutral amino acid transporter/retroviral receptor are translationally initiated at multiple alternative CUG and GUG codons

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.     

DNA sequence at the end of the cI gene in bacteriophage lambda.

The nucleotide sequence of 57 base pairs near the end of the cI gene in phage lambda is presented. This sequence was determined by direct sequencing techniques and includes the codons for 11 carboxyterminal aminoacids of the cI product, the lambda repressor. The sequence reveals that the cI gene, which has recently been shown to have a unique initiation region, is terminated by a UGA codon. A GUG triplet, which could act as a translation start signal for the rex gene occurs 8 base pairs beyond the cI termination codon. This GUG triplet is preceded by a sequence that could serve as a strong ribosome binding site for the rex message.     

Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.     

Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control.

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

tRNA hopping: enhancement by an expanded anticodon.

At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons. Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences. We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing. Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site. The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine). In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues. It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site.     

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.     

Limitations on the recombinant plasmid selection by Lac(+)/Lac(-) colony phenotype detection

Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids.     

Nucleotide sequence of turnip yellow mosaic virus coat protein mRNA

The primary structure of the coat protein messenger RNA of turnip yellow mosaic virus is presented. This sequence is the first complete nucleotide sequence of the coat protein messenger of a plant virus to be reported. The coding region, consisting of 567 nucleotides, is flanked by a 5′ noncoding region of 19 nucleotides (not including the initiation codon and the cap structure) and by a 3′ noncoding region of 109 nucleotides (including the termination signal). The coat protein mRNA has a base composition identical to that of the genome RNA with, in particular, the same high content in cytosine (38%). The codons that govern the incorporation of amino acids into the coat protein are nonrandomly utilized: >50% of the time the third base of the codons used is a cytosine. This pattern of codon preference is particularly marked for Leu, lie, Val, Thr and Cys.     

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.