The leader sequence of tobacco mosaic virus RNA devoid of Watson-Crick secondary structure possesses a cooperatively melted, compact conformation

The 5'-untranslated region (5'-UTR) of RNA of tobacco mosaic virus (TMV), called omega sequence, is known as an mRNA leader promoting efficient initiation of translation. The central part of the sequence consists of many CAA repeats, which were reported to be mainly responsible for the enhancing activity of the omega leader. In this work we synthesized the polyribonucleotides containing either the natural omega sequence or the regular (CAA)(n) sequence, and studied them using UV spectrophotometry and analytical ultracentrifugation methods. It was demonstrated that the polyribonucleotides manifest significant hypochromicity, cooperative melting of their structures upon heating, high melting temperature, and the sedimentation coefficients typical of compactly folded RNAs of this size. Thus, the omega leader and its core (CAA)(n) repeat sequence devoid of secondary structure of the Watson-Crick type seem to be well structured elements of mRNA.     

Mutational analysis of the tobacco mosaic virus 5''-leader for altered ability to enhance translation.

Mutational analysis of the 5'-untranslated leader sequence (omega) of tobacco mosaic virus (TMV) was carried out to determine those sequences necessary for the translational enhancement associated with omega. Five deletion mutants, a single base substitution, and a 25 base replacement mutant were tested for alterations in omega's ability to enhance expression of beta-glucuronidase (GUS) mRNA in tobacco mesophyll protoplasts and Escherichia coli or chloramphenicol acetyltransferase (CAT) mRNA in Xenopus laevis oocytes. Alteration of an eight base subsequence required for the binding of a second ribosome resulted in the loss of translational enhancement in X. laevis oocytes but not in protoplasts. Substantial increases in enhancement were observed for several of the mutants in E. coli.     

Insight into the structural organization of the omega leader of TMV RNA: the role of various regions of the sequence in the formation of a compact structure of the omega RNA

The 5′-untranslated sequence of tobacco mosaic virus RNA – the so called omega leader – is a well-known translational enhancer. The structure of the omega RNA has unusual features. Despite the absence of extensive secondary structure of the Watson–Crick type, the omega RNA possesses a stable compact conformation. The central part of the omega sequence contains many CAA repeats and is flanked by U-rich regions. In this work we synthesized the polyribonucleotides containing modified omega sequences, and studied them using analytical ultracentrifugation and thermal melting techniques. It was demonstrated that changes made in both the central and the 3′-proximal part of the sequence led to a strong destabilization of the omega RNA structure. We conclude that the regular (CAA)_n core region and the 3′-proximal AU-rich region of the omega RNA interact with each other and contribute together to the formation of a stable tertiary structure.     

Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA

The 5′-untranslated sequence of tobacco mosaic virus RNA — the so-called omega leader — exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson-Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson-Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson-Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)_n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson-Crick type.     

The highly repetitive region of the Helicobacter pylori CagY protein comprises tandem arrays of an alpha-helical repeat module

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagY_rpt2) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagY_rpt2 to comprise two highly thermostable and acid-stable α-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagY_rpt2 structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagY_rpt2 assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagY_rpt2 region.     

Leader length and secondary structure modulate mRNA function under conditions of stress.

Simian virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonucleotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.     

Correlation of Simple Sequence Repeat (SSR) Variants in the Leader Sequence of the waxy Gene with Amylose Content of the Grain in Rice

Variation of two simple sequence repeats (SSRs) in the leader region of the waxy gene was analyzed in a sample of 74 accessions, including Oryza sativa L. ssp. indica, japonica and wild rice ( O.rufipogon) representing a wide distribution range of amylose content (AC) in cultivated rice. Eight alleles were detected in the (CT)n motif and two alleles were resolved in the (AATT)n motif. The distribution of the alleles of the two SSRs was quite uneven as detected by the (CT)n motif. The repeat numbers of the two SSR motifs, (CT)n and (AATT)n, appeared to be inversely related such that the total length of this region was maintained. AC of the varieties was highly correlated with the length of SSRs. Differences in AC among the various SSR genotypes were statistically highly significant as analyzed using genotypes of both SSR motifs. Although the SSR variation did not seem to have obvious function in the synthesis of the starch synthase encoded by the waxy gene, the almost perfect correlation between the two SSRs and AC level could be used for quality improvement in rice breeding programs.     

水稻蜡质基因引导区的两个SSR序列与直链淀粉含量的相关性(英文)

分析了蜡质基因引导区的两个简单重复序列 (SSR) (CT) n 和 (AATT) n 在 74份水稻材料中的多态性及其与直链淀粉含量 (AC)的关系。这些材料包括了籼稻 (OryzasativaL .ssp .indica)、粳稻 (O .sativassp .japonica)和普通野生稻 (O .rufipogon) ,其AC值覆盖了栽培稻AC分布的整个范围。以 (CT) n 作标记检测到 8个等位基因 ,粳稻品种趋于含有重复数目较多 (n≥ 16 )的等位基因 ,重复次数较少 (n≤ 14)的等位基因只出现在籼稻中。 (AATT)n检测到 2个等位基因 ,野生稻中少数植株表现出杂合性。分析表明AC与这两个SSR序列基因型高度相关 ,高AC (>2 2 .0 % )品种具有 (CT)重复次数较少 (n≤ 14)的等位基因 ;相反 ,除了糯米外 ,所有低或者中等AC的品种都有 (CT)重复数较多 (n≥ 16 )的等位基因。具有重复次数较多的 (AATT) 6等位基因的品种多为高AC ,具有重复次数较少的(AATT) 5等位基因的品种多为低或中等AC。不同SSR基因型品种间AC差异极显著。虽然目前还不能确定这两个SSR序列在直链淀粉合成中的直接功能 ,SSR变异与AC间近乎完全的相关性可作为分子标记直接用于水稻的品质改良。     

Electron microscopic demonstration of the presence of amplified sequences at the 5''-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.     

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.     

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.