Functional evidence for D- and T-loop interactions in tmRNA

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.     

tmRNA

ｔｍＲＮＡ王德宝（中国科学院上海生物化学研究所,上海２０００３１）早在本世纪六十年代初期,就知道蛋白质的生物合成和三类ＲＮＡ有关。它们是ｔＲ－ＮＡ,ｍＲＮＡ和核糖体的ｒＲＮＡ。ＤＮＡ的蛋白质基因要先转录成ｍＲＮＡ,然后在核糖体上,ｍＲＮＡ的密码子和带...     

The tmRNA website

The tmRNA Website collects all available tmRNA sequences into a single public resource, along with alignments and a guide to searching for new sequences. Over the last year, several sequences have been updated or newly found by monitoring ongoing genome sequencing projects; tmRNA sequence data from 70 species are now available. New features include: color-coding of sequences to mark suggested base-paired regions, a list of the literature concerning tmRNA, careful crediting of tmRNA sequence identifications, and a split browser window. Updates are very frequent. The tmRNA Website has a new URL: http:www.indiana.edu/~tmrna     

The tmRNA Website.

tmRNA (also known as 10Sa RNA) is so-named for its dual tRNA-like and mRNA-like nature. It is employed in a remarkable trans -translation process to add a C-terminal peptide tag to the incomplete protein product of a broken mRNA; the tag targets the abnormal protein for proteolysis. tmRNA sequences have been identified in genomes of diverse bacterial phyla, including the most deeply branching. They have also been identified in plastids of the 'red' lineage. The tmRNA Website (http://www.wi.mit. edu/bartel/tmRNA/home ) contains a database currently including sequences from 37 species, with provisional alignments, as well as the tentatively predicted proteolysis tag sequences. A brief review and guide to the literature is also provided.     

Comparative sequence analysis of tmRNA

Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.     

tmRDB (tmRNA database)

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and accessible on the World Wide Web at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. Mirror sites are located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Institute of Biological Sciences, Aarhus, Denmark (http://www.bioinf.au. dk/tmRDB/). The tmRDB provides information and citation links about tmRNA, a molecule that combines functions of tRNA and mRNA in trans-translation. tmRNA is likely to be present in all bacteria and has been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana. This release adds 26 new sequences and corresponding predicted tmRNA-encoded tag peptides for a total of 86 tmRNAs, ordered alphabetically and phylogenetically. Secondary structures and three-dimensional models in PDB format for representative molecules are being made available. tmRNA alignments prove individual base pairs and are generated manually assisted by computational tools. The alignments with their corresponding structural annotation can be obtained in various formats, including a new column format designed to improve and simplify computational usability of the data.     

The tmRNA website.

tmRNA (10Sa RNA) has a central role in trans -translation, in which a peptide tag encoded in tmRNA is added to the abnormally short protein product of a broken mRNA, as a signal for proteolysis of the entire tagged protein. The tmRNA website was established in 1997 as a resource for phylogenetic considerations of tmRNA structure and function. Since then, three partial tmRNA sequences have been completed, and sequences from 13 more species have been identified. Forty-six species from 10 bacterial phyla and chloroplasts are now represented in the database. Provisional sequence alignments and predicted proteolysis tag sequences are provided, as well as a literature review and a guide to searching for new tmRNA sequences. The tmRNA website is accessible via WWW at a new URL: http://sunflower.bio.indiana.edu/kwilliam/tmRNA /home.html     

tmRDB (tmRNA database)

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Bioinformatics Research Center, Aarhus, Denmark (http://www.bioinf.au.dk/tmRDB/). The tmRDB collects and distributes information relevant to the study of tmRNA. In trans-translation, this molecule combines properties of tRNA and mRNA and binds several proteins to form the tmRNP. Related RNPs are likely to be functional in all bacteria. In this release of tmRDB, 186 new entries from 10 bacterial groups for a total of 274 tmRNA sequences have been added. Lists of the tmRNAs and the corresponding tmRNA-encoded tag-peptides are presented in alphabetical and phylogenetic order. The tmRNA sequences are aligned manually, assisted by computational tools, to determine base pairs supported by comparative sequence analysis. The tmRNA alignment, available in a variety of formats, provides the basis for the secondary and tertiary structure of each tmRNA molecule. Three-dimensional models of the tmRNAs and their associated proteins in PDB format give evidence for the recent progress that has been made in the understanding of tmRNP structure and function.     

tmRDB (tmRNA database)

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and is accessible on the WWW at URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. A tmRDB mirror site is located on the campus of Auburn University, Auburn, Alabama, reachable at the URL http://www.ag.auburn.edu/mirror/tmRDB/. Since April 1997, the tmRDB has provided sequences of tmRNA (previously called 10Sa RNA), a molecule present in most bacteria and some organelles. This release adds 17 new sequences for a total of 60 tmRNAs. Sequences and corresponding tmRNA-encoded tag peptides are tabulated in alphabetical and phylo-genetic order. The updated tmRNA alignment improves the secondary structures of known tmRNAs on the level of individual basepairs. tmRDB also provides an introduction to tmRNA function in trans-translation (with links to relevant literature), a limited number of tmRNA secondary structure diagrams, and numerous three-dimensional models generated interactively with the program ERNA-3D.     

Functional significance of an evolutionarily conserved alanine (GCA) resume codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.     

Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.     

Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.     

Comparative 3-D Modeling of tmRNA

Background

Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA.

Results

To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA.

Conclusion

Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.     

Functional interactions between nucleoporins and chromatin

As the gatekeepers of the eukaryotic cell nucleus, nuclear pore complexes (NPCs) mediate all molecular trafficking between the nucleoplasm and the cytoplasm. In recent years, transport-independent functions of NPC components, nucleoporins, have been identified including roles in chromatin organization and gene regulation. Here, we summarize our current view of the NPC as a dynamic hub for the integration of chromatin regulation and nuclear trafficking and discuss the functional interplay between nucleoporins and the nuclear genome.     

tmRNA的结构与功能

tmRNA是一类具有类似tRNA分子和mRNA分子双重功能的小分子RNA,它在一种特殊的翻译模式——反式翻译模式过程中发挥重要作用。最近又发现它与基因的表达调控及细胞周期的调控等生命过程密切相关。因此,关于tmRNA的研究已经引起了研究者的高度重视,它将是RNA组学研究的一个重要内容。文章主要论述了近年来关于tmRNA结构和功能方面的一些研究进展。Abstract: tmRNA is a dual small RNA similar to a tRNA and a mRNA which plays an important role in an unusual mode of translation——trans translation. Recently, it was found that tmRNA had something to do with regulating gene expression and cell cycle. So the researchers have paid much attention on such area. Furthermore, it is an important part in RNomics era. This article reviews the progress about the structure and function of tmRNA in recent years.     

Functional architecture of long-range perceptual interactions

The pattern of lateral interactions in the primary visual cortex, which has emerged from recent studies, conforms to the grouping rules of similarity, proximity, smoothness and closure. The goal of this paper is to understand the perceptual salience of oriented elements that are specifically organized to form a smooth contour. An overview of recent studies, in combination with new experimental results, is presented here to emphasis the idea that visual responses depend on input from both the center and the surround of the classical receptive field (CRF). It is assumed that normal lateral interactions produce a neuronal network that is formed by two antagonistic mechanisms: (i) excitation, that is spatially organized along the optimal orientation (collinear), and is predominant near the contrast threshold of the neuron, and (ii) inhibition, that is less selective and is distributed diffusely around the cell's response field. Thus, the inputs from the CRF and the anisotropic surround are summated non-linearly. The specificity of the facilitation and suppression along the collinear direction suggests the existence of second-order elongated collinear filters, which may increase the response similarity between neurons responding to elongated stimulus, thus may enhance the perceptual salience of anisotropic configurations such as contours. This causal connection is particularly evident in amblyopes, where abnormal development of the network results in the abnormal perception of contours.