Direct Monitoring of Initiation Factor Dynamics through Formation of 30S and 70S Translation–Initiation Complexes on a Quartz Crystal Microbalance

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S‐IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S‐IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S‐IC) and a 70S‐IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N‐terminally fused to biotin carboxyl carrier protein (bio‐BCCP), were immobilized on a Neutravidin‐covered QCM plate. By using bio‐BCCP‐IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S‐IC, and formation of the 70S‐IC. The kinetic parameters implied that the release of IF2 from the 70S‐IC could be the rate‐limiting step in translation initiation. The IF3‐immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet‐tRNA^fMet but did not lead to complete dissociation from the 30S‐IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S‐IC and 70S‐IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.     

Protein factors mediating selenoprotein synthesis

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.     

Genetic control by a metabolite binding mRNA

Messenger RNAs are typically thought of as passive carriers of genetic information that are acted upon by protein- or small RNA-regulatory factors and by ribosomes during the process of translation. We report that the 5'-untranslated sequence of the Escherichia coli btuB mRNA assumes a more proactive role in metabolic monitoring and genetic control. The mRNA serves as a metabolite-sensing genetic switch by selectively binding coenzyme B(12) without the need for proteins. This binding event establishes a distinct RNA structure that is likely to be responsible for inhibition of ribosome binding and consequent reduction in synthesis of the cobalamin transport protein BtuB. This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.     

Phosphorothioate Modification of mRNA Accelerates the Rate of Translation Initiation to Provide More Efficient Protein Synthesis

Messenger RNAs (mRNAs) with phosphorothioate modification (PS‐mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell‐free translation system. Protein synthesis from PS‐mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22‐fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′‐end to the initiation codon. Single‐turnover analysis of PS‐mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non‐natural mRNA.     

Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era

The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. This combination provides the cancer biologist with the ability to (i) identify the potential protein:protein associations and (ii) fully characterize function-critical post-translational modifications, both directly from silver-stained polyacrylamide gels. In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general. The first is the identification of gel-separated, low abundance proteins based on amino acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BclX(L). The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E-BP1, which controls mRNA translation.     

Post-transcriptional control of selenoprotein biosynthesis

Selenoproteins are defined as proteins containing the 21st proteinogenic amino acid, selenocysteine (Sec). Sec is encoded by UGA (STOP) codons which are re-coded to Sec by the presence of a selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region of selenoprotein mRNAs. The SECIS element is bound by several proteins, including SECIS-binding protein 2 (SBP2). Translation of selenoproteins critically depends on the integrity of the SECIS element - SBP2 interaction. Mutations in a SECIS element can abrogate expression of the respective selenoprotein. Mutations in SBP2 impinge on biosynthesis of a subset of selenoproteins and lead to a syndrome including hormonal, neurological, immunological symptoms as well as myopathy. Several other RNA-binding proteins are involved in selenoprotein translation and mediate the hierarchical response of selenoproteins to selenium deficiency. Global inhibition of selenoprotein translation is lethal in the mouse and hypomorphic mutations in selenocysteine synthase in humans leads to Progressive Cerebello Cerebral Atrophy, a neurodevelopmental and neurodegenerative disease in pediatric patients.     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

Translation of genetic information on the ribosome

The information for protein structure that is contained in the base sequence of the nucleic acids is translated on the ribosome into the amino acid sequence. This translation can be divided into chain initiation, chain growth, and chain termination. Several specific protein factors and nucleic acids are involved in each section.—For chain initiation, start complexes are formed from the initiating amino acyl-tRNA, mRNA carrying the start signal, and the small and large subunits of a ribosome. GTP and the initiating factors are also involved in this process.—In chain elongation, one amino acid at a time is transferred, in a reaction cycle, from the linkage with tRNA into a linkage with the polypeptide chain. The amino acid to be incorporated is initially bound to the ribosome as amino acyl-tRNA, a process for which GTP and protein factors are necessary. The subsequent formation of a peptide linkage is catalyzed by the peptidyl transferase of the large ribosomal subunit. The peptidyl-tRNA with its newly added amino acid residue is then transferred from the amino acyl-tRNA acceptor site A to the peptidyl donor site P of the ribosome. This requires another protein factor and cleavage of GTP into GDP and phosphate.–Ghain termination begins as soon as one of the three terminator triplets UAA, UAG, or UGA in the mRNA reaches the ribosome. The mRNA is moving in relation to the ribosome from the 5′ end to the 3′ end. Release of the completed polypeptide chain from the ribosome is dependent on release factors. Before initiation of a new polypeptide chain, the ribosomes dissociate into their subunits.     

A novel strategy by the action of ricin that connects phenotype and genotype without loss of the diversity of libraries.

We present a novel strategy for connection of phenotype and genotype in vitro that can be used for the selection of functional proteins even at room temperature. The strategy involves generation of a stable complex between a ribosome, an mRNA, and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. We demonstrate the potential selection capacity of this novel strategy by isolating such complexes that contain newly synthesized streptavidin and glutathione-S-transferase (GST) using appropriate ligands. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus our novel "Ribosome-Inactivation Display System (RIDS)" should provide, without loss of the pool population, a reliable, simple, and robust selection system for in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.     

Characterization of ribosomal proteins as biomarkers for matrix-assisted laser desorption/ionization mass spectral identification of Lactobacillus plantarum

For rapid identification of bacteria by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a bioinformatics approach using ribosomal subunit proteins as biomarkers has been proposed. This method compares the observed masses for biomarkers with calculated masses as predicted from the amino acid sequences registered on protein databases. To evaluate this approach, the expressed ribosomal proteins of a genome-sequenced bacterium, Lactobacillus plantarum NCIMB 8826, were characterized as a model sample. The protein expression of 42 ribosomal subunit proteins, together with 10 ribosome-associated proteins in the isolated ribosome fraction, was confirmed through two-dimensional gel electrophoresis combined with peptide mass fingerprinting. The observed masses of the proteins in the isolated ribosome fraction were then determined by MALDI-MS. We preliminarily selected 44 biomarkers whose observed masses were matched with the calculated masses predicted from the amino acid sequence registered in the protein databases by considering N-terminal methionine loss only. Of these, the finally selected reliable biomarkers were 34 proteins including 31 ribosomal subunit proteins and 3 ribosome-associated proteins that could be observed in the MALDI mass spectra of the cell lysate sample. These biomarkers were usable in MALDI-MS characterization of two industrial L. plantarum cultures.     

Direct monitoring kinetic studies of DNA polymerase reactions on a DNA-immobilized quartz-crystal microbalance

Catalytic reactions of DNA polymerase I from E. coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 10(8)M(-1) (k(on) = 10(5)M(-1)s(-1) and k(off)= 10(-3)s(-1)), and decreased to 10(6)M(-1) (k'on = 10(4)M(-1)s(-1) and k'off = 10(-2)s(-1)) for completely polymerized DNA. This is due to the 10-fold decrease in binding rate constant (k(on)) and 10-fold increase in dissociation rate constant (k(off)) for completed DNA strands. Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (k(cat) and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.     

Off-target activity of TNF-α inhibitors characterized by protein biochips

Tumor necrosis factor-alpha inhibitors are widely and successfully used to treat rheumatic diseases. However, significant side effects have been reported. To detect the potential off-target activities of such inhibitors we characterized two therapeutic antibodies (adalimumab, infliximab) and one receptor fusion protein (etanercept) on protein biochips (UNIchip AV-400) containing a printed serial dilution of tumor necrosis factor-alpha and about 384 different human proteins. Etanercept binds to ten proteins (affinity: 20-33% of tumor necrosis factor-alpha recognition), and six of these proteins are related to ribosomal proteins. Interestingly, adalimumab binds to the same six proteins related to ribosomal proteins (affinity: 12-18%) as well as to four proteins crucially involved in ribosomal protein synthesis. Alignment of protein sequences indicates no significant sequence homology between these ten proteins bound by the biological drugs with the highest off-target activities. Taken together, our in vitro results demonstrate that a significant number of proteins are recognized by tumor necrosis factor-alpha inhibitors and are related to ribosome biogenesis.     

In Vitro Evolution and Selection of Proteins: Ribosome Display for Larger Libraries

The cell-free production of large protein libraries can now be achieved with the ribosome display technique. The use of intact ribosomes (R) in the translation of a mRNA library enables the rapid selection of the generated proteins (P) according to their affinity to a given immobilized binding partner (B).     

A kinetic study of protein binding to ecabet sodium using quartz-crystal microbalance.

To define the mechanism of the protection by ecabet sodium of the gastric mucosa, the characteristics of protein binding of this drug were investigated using a quartz-crystal microbalance (QCM) method. The binding rate constants (kb) and the binding amounts (delta m) were obtained from time courses of the frequency decrease (mass increase) of the QCM. The binding constants to proteins of two ecabet analogues (G1, ecabet type and G2, non-ionic ecabet type) were dependent on the pH, leading to large kb values at the acidic region. Furthermore, the kb values of G1 with the addition of bovine serum albumin (BSA) and bovine serum fibrinogen (BSF) at the acid region were larger than those of G2. The difference in kb values between G1 and G2 for porcine gastric mucin (PGM) is hardly discernible. Ecabet seems to be more heavily distributed in the ulcerous areas than in the intact mucosa, judging from the large binding constants of this drug to BSA and BSF compared with those to PGM. It is suggested that ecabet is bound to proteins by hydrophobic interaction, moreover, the electrostatic interaction between this drug and proteins (BSA and BSF) occurs at acidic pH region. On account of these interactions, ecabet sodium seems to have a more protective effect on an ulcer at intraluminal acidity than sucralfate. Finally, QCM was found to be a useful technique for detecting quantitatively the time course of binding proteins with drug.     

Encodamers: unnatural peptide oligomers encoded in RNA

Conventional display libraries are generally limited to the 20 naturally occurring amino acids. Here, we demonstrate that novel unnatural amide-linked oligomers can be constructed and encoded in an attached RNA for the purpose of mRNA display library design. To do this, we translated templates of various lengths in a protein synthesis system modified to promote sense codon suppression. Unnatural residues were escorted to the ribosome as chemically acylated tRNAs added to the translation mixture. Our experiments reveal that unnatural peptide oligomers ("encodamers") consisting of an N-substituted amino acid are readily generated as mRNA-peptide fusions with excellent stepwise efficiency. The N-substituted polyamides have strikingly improved proteolytic stability relative to their naturally encoded counterparts. Overall, our work indicates that the ribosome can be used as a synthesis platform to generate encoded combinatorial chemistry outside the universal genetic code.     

Is tRNA binding or tRNA mimicry mandatory for translation factors?

tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.