Expression and membrane integration of SARS-CoV M protein

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46–68 and 78–100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14–36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bifunctional transfer-messenger RNA

Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. This concerted reaction, known as trans-translation, contributes to translational quality control and regulation of gene expression in bacteria. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. Mutants lacking tmRNA activity typically have severe phenotypes, including defects in viability, virulence, and responses to environmental stresses.     

Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system

A protein-synthesizing system based on a minimal set of purified components was used to investigate the roles molecular chaperones play in the folding of newly synthesized polypeptides. After we ascertained that this system lacks intrinsic chaperones, the effect of adding chaperones in a co-translational or post-translational manner was directly evaluated. An aggregation-prone single-chain antibody was used as the model nascent chain. The participation of the trigger factor or the DnaK system during translation efficiently increased the level of functional protein that was generated. In addition, both systems also acted as chaperones after translation had been stopped. In contrast, the GroEL/ES system showed little or no co- or post-translational assistance in folding.     

寡核苷酸探针(CAC)5/(GTG) 5指纹的非放射性标记和检测

在ＤＮＡ指纹分析中,通常采用的是同位素３２Ｐ标记ＤＮＡ分子探针,可检测到微微克（ｐｇ）靶序列。但３２Ｐ半衰期很短（仅有１４天）,使用和运输均不方便,３２Ｐ还放出硬射线,对实验者身体造成损害,需要一定的防护措施,并且容易污染环境。因此,科学家们研制出了...     

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it''s now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.     

Low Complexity Regions behave as tRNA sponges to help co-translational folding of plasmodial proteins

In most organisms, the information necessary to specify the native 3D-structures of proteins is encoded in the corresponding mRNA sequences. Translational accuracy and efficiency are coupled and sequences that are slowly translated play an essential role in the concomitant folding of protein domains. Here, we suggest that the well-known mechanisms for the regulation of translational efficiency, which involves mRNA structure and/or asymmetric tRNA abundance, do not apply to all organisms. We propose that Plasmodium, the parasite responsible for malaria, uses an alternative strategy to slow down ribosomal speed and avoid multidomain protein misfolding during translation. In our model, the abundant Low Complexity Regions present in Plasmodium proteins replace the codon preferences, which influence the assembly of protein secondary structures.     

A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER

Sdh3/Shh3, a subunit of mitochondrial succinate dehydrogenase, contains transmembrane domains with a hydrophobicity comparable to that of endoplasmic reticulum (ER) proteins. Here, we show that a C-terminal reporter fusion to Sdh3/Shh3 results in partial mis-targeting of the protein to the ER. This mis-targeting is mediated by the signal recognition particle (SRP) and depends on the length of the C-terminal tail. These results imply that if nuclear-encoded mitochondrial proteins contain strongly hydrophobic transmembrane domains and a long C-terminal tail, they have the potential to be recognized by SRP and mis-targeted to the ER.     

Birth, life and death of nascent polypeptide chains

The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome ("birth") to full function ("maturity") involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation ("death"). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains.     

Predicting N-terminal acetylation based on feature selection method

Methionine aminopeptidase and N-terminal acetyltransferase are two enzymes that contribute most to the N-terminal acetylation, which has long been recognized as a frequent and important kind of co-translational modifications [R.A. Bradshaw, W.W. Brickey, K.W. Walker, N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families, Trends Biochem. Sci. 23 (1998) 263-267]. The combined action of these two enzymes leads to two types of N-terminal acetylated proteins that are with/without the initiator methionine after the N-terminal acetylation. To accurately predict these two types of N-terminal acetylation, a new method based on feature selection has been developed. 1047 N-terminal acetylated and non-acetylated decapeptides retrieved from Swiss-Prot database (http://cn.expasy.org) are encoded into feature vectors by amino acid properties collected in Amino Acid Index database (http://www.genome.jp/aaindex). The Maximum Relevance Minimum Redundancy method (mRMR) combining with Incremental Feature Selection (IFS) and Feature Forward Selection (FFS) is then applied to extract informative features. Nearest Neighbor Algorithm (NNA) is used to build prediction models. Tested by Jackknife Cross-Validation, the correct rate of predictors reach 91.34% and 75.49% for each type, which are both better than that of 84.41% and 62.99% acquired by using motif methods [S. Huang, R.C. Elliott, P.S. Liu, R.K. Koduri, J.L. Weickmann, J.H. Lee, L.C. Blair, P. Ghosh-Dastidar, R.A. Bradshaw, K.M. Bryan, et al., Specificity of cotranslational amino-terminal processing of proteins in yeast, Biochemistry 26 (1987) 8242-8246; R. Yamada, R.A. Bradshaw, Rat liver polysome N alpha-acetyltransferase: substrate specificity, Biochemistry 30 (1991) 1017-1021]. Furthermore, the analysis of the informative features indicates that at least six downstream residues might have effect on the rules that guide the N-terminal acetylation, besides the penultimate residue. The software is available upon request.     

The incoherence of the vesicle theory of protein secretion

The rates at which cells secrete peptides and proteins must on average equal their rate of synthesis. This basic equality has unanticipated and seemingly categorical negative consequences for the vesicle theory of protein secretion. This is because the transport mechanisms it proposes, such as the budding and fusion of small vesicles and secretion by exocytosis, are not capable of balancing forces. What follows is an account of the analysis that leads to this conclusion.