scholarly journals Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries

2018 ◽  
Vol 1 (5) ◽  
pp. e201800148 ◽  
Author(s):  
Britta Seip ◽  
Guénaël Sacheau ◽  
Denis Dupuy ◽  
C Axel Innis
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Throughput ◽  
Quantitative Measure ◽  
Ribosome Profiling ◽  
Coding Region ◽  
Versatile Tool

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-boundEscherichia coliribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.

scholarly journals High-throughput inverse toeprinting reveals the complete sequence dependence of ribosome-targeting antibiotics

2018 ◽  
Author(s):  
Britta Seip ◽  
Guénaёl Sacheau ◽  
Denis Dupuy ◽  
C. Axel Innis
Keyword(s):  
High Throughput ◽  
Quantitative Measure ◽  
Complete Sequence ◽  
Ribosome Profiling ◽  
Sequence Specificity ◽  
Messenger Rnas ◽  
Coding Region ◽  
Sequence Dependence

It has recently become clear that various antibiotics block the translation of bacterial proteins in a sequence-specific manner. In order to understand how this specificity contributes to antibiotic potency and select better antimicrobial leads, new high-throughput tools are needed. Here, we present inverse toeprinting, a new method to map the position of ribosomes arrested on messenger RNAs during in vitro translation. Unlike ribosome profiling, our method protects the entire coding region upstream of a stalled ribosome, making it possible to work with transcript libraries that randomly sample the sequence space. We used inverse toeprinting to characterize the pausing landscape of free and drug-bound bacterial ribosomes engaged in translation. We obtained a comprehensive list of arrest motifs that could be validated in vivo, along with a quantitative measure of their pause strength. Thus, our method provides a highly parallel and scalable means to characterize the sequence specificity of translation inhibitors.

scholarly journals Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

scholarly journals Primary Structure of Human Platelet Factor 4.

1977 ◽  
Author(s):  
F.J. Morgan ◽  
G.S. Begg ◽  
C.N. Chesterman
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Information ◽  
Human Platelet ◽  
Specific Protein ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Tryptic Peptides

The amino acid sequence of human platelet factor 4 (PF4) has been studied. PF4 is a platelet specific protein with antiheparin activity, released from platelets as a proteoglycan complex, whose measurement may provide an important index of platelet activation both in vivo and in vitro. These studies were undertaken to characterize fully the PF4 molecule. PF4 is a stable tetramer, composed of identical subunits, each with a molecular weight based on the sequence studies of approx. 7,770. Each PF4 subunit contains 69 amino acids, including 4 half-cystine (# 10, 12, 36, 37), one tyrosine (# 59), 3 arginine and 8 lysine, but no methionine, phenylalanine or tryptophan residues. The basic residues are predominantly in the C-terminal region. The tryptic peptides were aligned after studies which included tryptic digestion of citraconylated RCM-PF4, and automated Edman degradation of RCM-PF4 and citraconylated tryptic peptides. No glycopeptides were detected. This structural information should enable clear distinction to be made between PF4 and other platelet proteins such as β thromboglobulin. The provisional amino acid sequence of each subunit is:Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Cys-Pro-Thr-Ala-Gln-Ile-Leu-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Pro-Leu-Asp-Leu-Gln-Ala-Tyr-Leu-Lys-Ile-Lys(Lys, Lys, Ser, Glx, Leu, Leu)

scholarly journals Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

2016 ◽  
Vol 113 (7) ◽  
pp. E829-E838 ◽  
Author(s):  
Yuhei Chadani ◽  
Tatsuya Niwa ◽  
Shinobu Chiba ◽  
Hideki Taguchi ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Ribosome Profiling ◽  
Cytosolic Proteins ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Chemical Trait

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study,Escherichia coliSecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members ofE.colithat were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

scholarly journals Innovative HBV Animal Models Based on the Entry Receptor NTCP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 828 ◽  
Author(s):  
Jochen M. Wettengel ◽  
Benjamin J. Burwitz
Keyword(s):  
Amino Acid ◽  
Animal Models ◽  
Hepatitis B ◽  
Amino Acid Sequence ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Species Barrier ◽  
Bile Acid Transporter

Hepatitis B is a major global health problem, with an estimated 257 million chronically infected patients and almost 1 million deaths per year. The causative agent is hepatitis B virus (HBV), a small, enveloped, partially double-stranded DNA virus. HBV has a strict species specificity, naturally infecting only humans and chimpanzees. Sodium taurocholate co-transporting polypeptide (NTCP), a bile acid transporter expressed on hepatocytes, has been shown to be one of the key factors in HBV infection, playing a crucial role in the HBV entry process in vitro and in vivo. Variations in the amino acid sequence of NTCP can inhibit HBV infection and, therefore, contributes, in part, to the species barrier. This discovery has revolutionized the search for novel animal models of HBV. Indeed, it was recently shown that variations in the amino acid sequence of NTCP represent the sole species barrier for HBV infection in macaques. Here, we review what is known about HBV entry through the NTCP receptor and highlight how this knowledge has been harnessed to build new animal models for the study of HBV pathogenesis and curative therapies.

scholarly journals Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin

1998 ◽  
Vol 334 (3) ◽  
pp. 659-667 ◽  
Author(s):  
Christine RASCHER ◽  
Andreas PAHL ◽  
Anja PECHT ◽  
Kay BRUNE ◽  
Werner SOLBACH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Isomerase Activity ◽  
Terminal Amino ◽  
Pathogenic Protozoan ◽  
Parasite Leishmania

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA–Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

scholarly journals Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

2002 ◽  
Vol 184 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
In Vitro Transcription ◽  
Dna Binding Domain ◽  
Promoter Activation ◽  
Transcription Activators

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

scholarly journals In-vitro and In-silico approach for characterization of antimicrobial-peptide from probiotics against Staphylococcus aureus and Escherichia coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmidevi N
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The present aim was to determine the characteristic feature and stability of antimicrobial compound (AMC) produced by probiotic strains of Enterococcus durans MCC4243, Lactiplantibacillus plantarum (Basanym: Lactobacillus plantarum MCC4246) and Limosilactobacillus fermentum (Basonym: Lactobacillus fermentum MCC4233) against Staphylococcus aureus MTCC96 and Escherichia coli MTCC118. Growth kinetic assay revealed 24h of incubation to be optimum for bacteriocin production. Ammonium sulphate precipitation-dialysis was found to be favorable method for extraction of AMC compared to other methods employed. The partially purified compound after ion-exchange chromatography was found to be thermo-resistant upto 90°C and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. The apparent molecular weight of AMC from MCC4243 and MCC4246 was found to be 3.5KDa. PCR confirmed the presence of plantaricinA gene in MCC4246. Translated partial amino acid sequence of plnA gene of MCC4246 displayed 48 amino acid sequence which had 100% similarity with plantaricinA of Lactobacillus plantarum (WP 0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns and functions on cytoplasm with 10.82 isoelectric point and 48.6% hydrophobicity. From the study, the amino acid sequence “KSSAYSLQMGATAIKQVKKLFKKWGW” of peptide was predicted to be responsible for antimicrobial activity.

scholarly journals Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Translation Initiation ◽  
Single Gene ◽  
Ribosome Profiling ◽  
Genome Wide ◽  
Translation Start ◽  
Alternative Translation

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

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