Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25.Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.     

Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.     

RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69

The DNA polymerases (gp43s) of the two related phages T4 and RB69 are DNA-binding proteins that also function as mRNA-binding autogenous translational repressors. As repressors, T4 gp43 is narrowly specific to its own mRNA whereas RB69 gp43 is equally effective against mRNA for either protein. We used in vitro RNase-sensitivity and RNA footprinting assays to identify features of the non-identical T4 and RB69 mRNA targets (translational operators) that allow for their identical binding affinities and biological responses to RB69 gp43. We observed that T4 gp43 and RB69 gp43 produce identical footprints on RNA substrates bearing the T4-derived operator, suggesting that the two gp43s make identical contacts with this operator. In contrast, the footprint produced by RB69 gp43 on its autogenous RNA target was shorter than its footprint on operator RNA from T4. As expected, we also observed only weak protection of RB69-derived operator RNA from RNase by T4 gp43; however, photocross-linking studies suggested that T4 gp43 recognizes structural features of the RB69-derived operator that are not detected by RNase- sensitivity assays. The results suggest that RB69 gp43 and T4 gp43 differ in their abilities to use RNA-sequence-independent interactions to configure potential RNA targets for translational repression.     

Efficiency and Frequency of Translational Coupling between the Bacteriophage T4 Clamp Loader Genes

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the “sliding clamp” of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the “clamp loader” activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes 44 and 62, which are directly adjacent on polycistronic mRNA molecules, are—to some degree—translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ~85% and a coupling frequency of ~25% between the 44-62 gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.     

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.     

Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.     

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.     

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.     

Y-box Binding Protein 1: Providing a New Angle on Translational Regulation

Current models of translational regulation are mostly focused on how translational factors engage a messenger mRNA to the ribosome to initiate translation. Since the majority of mRNAs in eukaryotes are translated in a cap-dependent manner, the mRNA 5’ cap-binding protein eIF4E was characterized as a key player responsible for the recruitment of mRNAs to the initiation complex. The availability of eIF4E is believed to be especially critical for translational activation of mRNAs with extensive secondary structures in their 5’UTRs, many of which code for labile regulatory proteins essential for cell growth or viability. Surprisingly, little attention is paid to the other side of translational control, e.g., to define mechanisms responsible for translational silencing and storage of the above messages. In this review, we discuss the possibility that eIF4E per se may not be sufficient to release mRNAs from translational block. We found that many growth- and stress-related mRNAs are associated with the translational repressor YB-1, which can compete with the eIF4E-driven translation initiation complex for binding to the capped 5’ mRNA terminus. Moreover, the cap-dependent repressor activity of YB-1 appears to be negatively regulated via Akt-mediated phosphorylation of the Ser-102 residue of YB-1. Taken together with recent evidence suggesting that translational activation of growth-related messages is a primary cellular response to activation of Ras-Erk and PI3K-Akt signaling pathways, our data suggest that differential expression of specific mRNA subsets is regulated by the PI3K-Akt pathway and achieved via coordinated activation of the components of translational machinery and inactivation of general translational repressors such as YB-1.     

Lack of Discrimination between Early and Late T4 mRNA by Host Initiation Factors

DURING development of T4 phage in E. coli, control at the translational level may play an important part in switching the reading of early to late T4 messenger RNAs. In vitro experiments have shown that protein factors isolated from ribosomes of T4 infected cells can restrict translation of either host mRNA or R17 phage RNA, whilst permitting normal translation of late T4 mRNA^1–4. This alteration of specificity has been attributed to an initiation factor F3^5,6. It is unlikely that this switch is related to the shut-off of host protein synthesis which occurs immediately after infection^7,8, because they occur at distinctly different times in vivo^{1, 3}.     

原核生物小RNA的研究及其进展

原核生物中的小RNA（small RNA,sRNA）长度通常在50—250nt之间,一般在细胞内不被翻译,对基因转录后水平的调控发挥着关键作用。最初在大肠杆菌中发现,通过计算机预测和实验技术分析,查明的种类现已近140种,其作用机制包括;与目标mRNA的翻译起始位点或前导链结合分别抑制或促进翻译;或者模拟其他核酸的二级结构,去除mRNA结合蛋白对翻译的阻抑作用,促进翻译。此外,在转录水平上,SRNA还能模拟开放的启动子结构与RNA聚合酶结合阻止转录。     

Autogenous Regulation of the Rega Gene of Bacteriophage T4: Derepression of Translation

The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.     

Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.