On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.     

乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的小鼠免疫应答研究

目的 构建乙型肝炎病毒核心抗原(HBcAg)和Flt3配体(FL)胞外段双表达核酸疫苗,并观察其免疫原性。方法 分别将HBcAg、FL基因克隆入pJW4303载体,获得双表达核酸疫苗,体外转染293T细胞检测目的基因的表达。分组免疫BABL／c小鼠,酶联免疫吸附试验(ELISA)检测小鼠血清抗-HBc IgG效价,酶联免疫斑点试验(ELISPOT)检测HBcAg特异性Th1／Th2型细胞因子的分泌水平。结果 所构建疫苗在体外均能表达HBcAg和FL,当基因位于内部核糖体切入位点(IRES)元件上游时表达水平明显较优。pJW4303／C／FL免疫组产生的抗-HBc IgG效价和Th1／Th2型细胞因子的分泌水平均显著优于pJW4303／C和pJW4303／FL／C组。结论 成功构建双表达核酸疫苗,基因位于上游时表达水平高于下游。FL基因的引入明显增强了HBcAg核酸疫苗的免疫原性。     

IS THE GROWTH RATE HYPOTHESIS APPLICABLE TO MICROALGAE?1

The growth rate hypothesis (GRH) asserts, from known biochemistry, that maintaining high growth rates requires high concentrations of ribosomes. Since ribosomes are rich in phosphorus (P), the GRH predicts a positive correlation between growth rate and P content; this correlation is observed in some organisms. We consider the application of the GRH to phytoplankton and identify several key problems that require further research before the hypothesis can be accepted for these organisms. There are severe methodological problems that confound interpretation of data for testing the GRH. These problems include the measurement of protein and nucleic acids (such that ratio of these components carries a high level of uncertainty), studies of steady‐state versus dynamic systems, and the presentation of data per cell (especially as cell size varies with growth rate limitations) and the calculation of growth rates. In addition, because of the short generation times and rapid responses of these organisms to perturbations, ribosome and RNA content is expected to vary in response to (de)repression of various systems; content may increase on application of growth‐limiting stress. Finally, that most phytoplankton accumulate P when not P stressed conflicts with the GRH. In consequence, the value of the GRH for any sort of predictive role in nature appears to be severely limited. We conclude that the GRH cannot be assumed to apply to phytoplankton taxa without first performing experimental tests under transient conditions.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Mechanistic Role of Structurally Dynamic Regions in Dicistroviridae IGR IRESs

Dicistroviridae intergenic region (IGR) internal ribosome entry site(s) (IRES) RNAs drive a cap-independent pathway of translation initiation, recruiting both small and large ribosomal subunits to viral RNA without the use of any canonical translation initiation factors. This ability is conferred by the folded three-dimensional structure of the IRES RNA, which has been solved by X-ray crystallography. Here, we report the chemical probing of Plautia stali intestine virus IGR IRES in the unbound form, in the 40S-subunit-bound form, and in the 80S-ribosome-bound form. The results, when combined with an analysis of crystal structures, suggest that parts of the IRES RNA change structure as the preinitiation complex forms. Using mutagenesis coupled with native gel electrophoresis, preinitiation complex assembly assays, and translation initiation assays, we show that these potentially structurally dynamic elements of the IRES are involved in different steps in the pathway of ribosome recruitment and translation initiation. Like tRNAs, it appears that the IGR IRES undergoes local structural changes that are coordinated with structural changes in the ribosome, and these are critical for the IRES mechanism of action.     

The Efficiency of Selenocysteine Incorporation Is Regulated by Translation Initiation Factors

Selenocysteine (Sec) incorporation is an essential process required for the production of at least 25 human selenoproteins. This unique amino acid is co-translationally incorporated at specific UGA codons that normally serve as termination signals. Recoding from stop to Sec involves a cis-acting Sec insertion sequence element in the 3′ untranslated region of selenoprotein mRNAs as well as Sec insertion sequence binding protein 2, Sec-tRNA^Sec, and the Sec-specific elongation factor, eEFSec. The interplay between recoding and termination at Sec codons has served as a focal point in researching the mechanism of Sec insertion, but the role of translation initiation has not been addressed. In this report, we show that the cricket paralysis virus intergenic internal ribosome entry site is able to support Sec incorporation, thus providing evidence that the canonical functions of translation initiation factors are not required. Additionally, we show that neither a 5′ cap nor a 3′ poly(A) tail enhances Sec incorporation. Interestingly, however, the presence of the internal ribosome entry site significantly decreases Sec incorporation efficiency, suggesting a role for translation initiation in regulating the efficiency of UGA recoding.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

N-terminal deletion affects catalytic activity of saporin toxin

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated proteins with deletions at the N-terminus and at the C-terminus. An N-terminal (sapDelta1-20) deletion mutant of saporin displayed defective catalytic activity, drastically reduced cytotoxicity but increased ability to interact with liposomes inducing their permeabilization at low pH. A C-terminal (sapDelta239-253) deletion mutant showed instead a moderate reduction in cytotoxic activity. A substantial alteration of secondary structure was evidenced by Fourier transformed infrared spectroscopy (FTIR) in the sapDelta1-20 mutant. It can be hypothesized that the defective functions of sapDelta1-20 are due to alterations of its spatial configuration.     

GTPases of the prokaryotic translation apparatus

Bacterial protein synthesis involves four protein factors that belong to the GTPase family: IF2, EF-G, EF-Tu, and RF3. Their role in translation has attracted considerable interest over the recent decades. Cryoelectron microscopy has made it possible to monitor the dynamics of the ribosome upon binding of the translation factors, and biochemical findings have associated the structural data with functional changes in GTPases: the exchange of GDP for GTP, activation of GTPase, and changes in its conformation. The results have been used to construct models of GTPase action during prokaryotic translation. Data are accumulating that the ribosome simultaneously acts as a GDP/GTP exchange factor and a GTPase-activating factor for RF3, IF2, and EF-G. The review systematizes the most important experimental findings and theoretical models proposed for regulation of the functional cycle of prokaryotic translation GTPases.     

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.