An efficient system for small protein expression and refolding

The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin alpha (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins.     

Translation of the Chloroplast psbA mRNA Requires the Nuclear-encoded Poly(A)-binding Protein,RB47

A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5′ untranslated region of the psbA mRNA. The results presented here confirm RB47''s role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.     

Enhanced NMR signal detection of imino protons in RNA molecules containing 3′ dangling nucleotides

We present a method for improving the quality of nuclear magnetic resonance (NMR) spectra involving exchangeable protons near the base of the stem of RNA hairpin molecules. NMR spectra of five different RNA hairpins were compared. These hairpins consisted of a native RNA structure and four molecules each having different unpaired, or dangling, nucleotides at the 3′ end. NMR experiments were acquired in water for each construct and the quality of the imino proton spectral regions were examined. The imino resonances near the base of the stem of the wild type RNA structure were not observed due to breathing motions. However, a significant increase in spectral quality for molecules with dangling 3′ adenosine or guanosine nucleotides was observed, with imino protons detected in these constructs that were not observed in the wild type construct. A modest improvement in spectral quality was seen for the construct with a 3′ unpaired uridine, whereas no significant improvement was observed for a 3′ unpaired cytidine. This improvement in NMR spectral quality mirrors the increased thermodynamic stability observed for 3′ unpaired nucleotides which is dependant on the stacking interactions of these nucleotides against the base of the stem. The use of a dangling 3′ adenosine nucleotide represents an easy method to significantly improve the quality of NMR spectra of RNA molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The ‘DEAD/H’ family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast (Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are >95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.     

Folding transitions during assembly of the eukaryotic mRNA cap-binding complex

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.     

Lack of secondary structure characterizes the 5' ends of mammalian mitochondrial mRNAs

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5' and 3' untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5' ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than -3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5' ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5' sequences into the mRNA entrance site to participate in translation initiation.     

mRNA翻译起始区二级结构改变对干扰素基因在大肠杆菌中翻译的影响

为研究mRNA翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变α8干扰素及αA干扰素衍生物基因的5′端若干位点,使其与表达载体重组后转录形成的mRNA翻译起始区结构发生改变。SDS-PAGE及活性测定证实这些改变提高了外源基因的表达水平。RNA斑点印迹表明突变前后基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高。mRNA翻译起始区二级结构预测提示其生成自由能(ΔG)的变化可能与表达水平的提高有关。     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq

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