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Optical Tweezers for Manipulation of Single Molecules
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
Optical tweezers have also been used to probe the motion and mechanisms of nucleic acids motors such as the DNA packaging motor, RNA polymerase, and ribosome. The machinery involved in the packaging of viral DNA has two components, the portal-connector and ATPase. Through systematic studies of the packaging of viral DNA into the phi29 bacteriophage, Bustamante et al. demonstrated that the rotary portal motor of the bacteriophage can package DNA against high forces, and elucidated a minimal kinetic model of force generation [86–90]. RNA polymerase is the enzyme responsible for copying the information stored in a DNA sequence into the messenger RNA during the process of transcription. Using the high-resolution optical tweezers with single base-pair resolution, the groups of Bustamante and Block systematically studied the individual transcription events, characterized their heterogeneity, revealed their stochastic alternation in periods of continuous translocation and pauses, and provided kinetics models of chemo-mechanical coupling in transcription [91–101]. Ribosomes are complex molecular machines that hydrolyze GTP to translate the information encoded in mRNA into proteins. During protein biosynthesis, the ribosome moves along the mRNA in the 5′-to-3′ direction, catalyzed by the forward translocase elongation factor G (EF-G). Through subtle design, the group led by Tinoco and Bustamante successfully used optical tweezers to study translation by a single ribosome on one mRNA (Figure 2.8) [102–110]. They showed that translation of a single messenger RNA by a ribosome occurs by successive translocation-and-pause cycles. They further studied the modulation of nascent protein folding by the ribosomal environment, and provided a kinetic model describing how a protein can regulate its own synthesis by the force generated during folding [107]. Through direct measurement of ribosome-dependent mRNA dynamics during programmed frameshifting, they demonstrated multiple ribosomal translocation attempts while in register with a slippery sequence [108].
Biocatalysis: An introduction
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
The sense strand is first transcribed into a messenger RNA (mRNA; RNA contains A, G, C, and uracil (U)) catalyzed by DNA-dependent RNA polymerases in presence of ribonucleoside triphosphates before the information is translated into the respective amino acid sequence constituting the primary structure of the polypeptide chain. The site of the protein biosynthesis, always starting with (N-formyl) methionine (fMet or Met), is the ribosome, a protein/RNA complex. Translation proceeds by linking amino acids to their respective transfer RNAs (tRNA) for transfer to the ribosome. tRNA in its three-dimensional compact structure resembles an “L” containing four unpaired loops. As a consequence of the L-like tertiary structure, the molecule is dipolar. One pole is the anticodon in a variable loop; the three nucleobases of the anticodon are complementary to an amino acid codon of the mRNA. The second pole is located at a distance of about 8 nm from the anticodon. It is characterized by the invariant sequence 5’-CCA-3’ identical in all tRNAs, and serves to bind the respective aa. Aminoacyl-tRNA-synthetases (AARSs) charge the different tRNAs in an ATP-dependent reaction. The AARSs are specific for one amino acid each, and for the corresponding tRNA. The latter is recognized by the synthetase mainly via characteristic structural elements near to the anticodon of the tRNA, and its acceptor 3’- end, respectively. Linking the different activated amino acids via peptide bond formation occurs at the ribosome consisting of a small 30 S subunit and a large 50 S subunit (prokaryotic ribosome; S: Svedberg units). The small subunit made up by about 20 proteins serves to stabilize the position of the mRNA and a 16S-rRNA. This rRNA recognizes the so-called Shine-Delgarno sequence (AGGAGGU) of an mRNA and supports mRNA binding by complimentary base pairing. The initiation phase is followed by the elongation process. The large subunit, possessing peptidyl transferase (PT) activity, has two binding sites, the P (peptide) and the A (acceptor) site, with tRNAfMet (tRNAMet) bound to the P-site. The elongation process requires elongation factors (EF) and starts with binding the second tRNA—matching the next codon of the mRNA—at the A-site. After the second aa residue has been transferred to the initiation aa fMet, tRNAfMet is released and the mRNA with the bound second tRNA moves from the A- to the P-site so that the next aminoacyl-tRNA can bind. This process is continued until the A-site presents one of the stop codons having no complimentary tRNAs. Under these conditions, releasing factors come into action and catalyze the final step, the hydrolysis of the ester bond between the last tRNA and the C-terminus of the peptide chain. A third site—the E (exit)-site is required for releasing the deacetylated tRNAs after transferring its amino acids to the growing peptide chain.
Green formulation, characterization, antifungal and biological safety evaluation of terbinafine HCl niosomes and niosomal gels manufactured by eco-friendly green method
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Katayoun Morteza-Semnani, Majid Saeedi, Jafar Akbari, Shakiba Hedayati, Seyyed Mohammad Hassan Hashemi, Seyyed Mobin Rahimnia, Amirhossein Babaei, Mona Ghazanfari, Iman Haghani, Mohammad Taghi Hedayati
A total of 64 molecularly identified isolates of Aspergillus (n = 44), Fusarium (n = 10) and Trichophyton (n = 10) were obtained from the collection of Invasive Fungi Research Center (IFRC) of Mazandaran University of Medical Sciences were included in the study. Identification of Aspergillus and Fusarium at species level were done by applying of sequencing of the β-tubulin-encoding gene (benA) and partial translation elongation factor 1 alpha (TEF-1alpha) gene, respectively. Trichophyton mentagrophytes was identified by squalene epoxidase gene (SQLE) locus. The clinical source of Aspergillus species including A. fumigatus and A. niger were from Broncho-alveolar lavage (BAL) of patients with different underlying conditions and Fusarium species from patients with onychomycosis. All Trichophyton mentagrophytes were originated from patients with confirmed cases of dermatophytosis who were clinical TER resistant according to our previous study (unpublished data).
An efficient method for recombinant production of human alpha synuclein in Escherichia coli using thioredoxin as a fusion partner
Published in Preparative Biochemistry & Biotechnology, 2020
Babak Saffari, Mehriar Amininasab, Sara Sheikhi, Jamshid Davoodi
During the standard osmotic shock procedure, bacterial cells are initially suspended in a hyperosmotic sucrose solution supplemented with EDTA. The concentrated sucrose solution causes the cells to shrink and EDTA contributes to the removal of lipopolysaccharide from the cell envelope, resulting in an enhanced permeability of the outer membrane. In the next step, cells are transferred into a cold hypotonic solution which leads to rapid cell swelling and subsequent release of periplasmic and some cytoplasmic proteins including the molecular chaperone DnaK, translational elongation factor Tu (EF-Tu), and trx[59–62]. The exact pathway by which a specific group of cytoplasmic proteins reach the periplasm in the absence of cell lysis is not yet fully understood. Nevertheless, two different possible mechanisms have been proposed in the literature, one via the large-conductance mechanosensitive channel (MscL)[59–60] and the other by means of a molecular sieve that is presumably formed by the transiently damaged bacterial envelope and allows the passage of proteins with a molecular mass of up to 100–150 kDa.[63]
Detection of multidrug resistant environmental isolates of acinetobacter and Stenotrophomonas maltophilia: a possible threat for community acquired infections?
Published in Journal of Environmental Science and Health, Part A, 2020
Reshme Govender, Isaac D Amoah, Sheena Kumari, Faizal Bux, Thor A Stenström
PCR was used to amplify and detect the presence of specific conserved sequences in presumptive S. maltophilia and Acinetobacter isolates using specific primers (Inqaba Biotech, SA) as listed in Table 2. DNA was extracted using the boiling method [58] and amplified with a T100 thermocycler (Bio-Rad, USA). The translation elongation factor P gene (efp) was targeted for the genus specific identification of Acinetobacter using the following cycling conditions; initial denaturation for 4 min at 94 °C, and 35 cycles of amplification, each for 45 s at 95 °C, 45 s at 52 °C, and 90 s at 72 °C and the final extension at 72 °C for 10 minutes.[49]Acinetobacter baumannii, ATCC 19606 was used as a positive control for the PCR assay. The identification of S. maltophilia was based on a 278 bp fragment of the 23S rRNA gene. The 23S rRNA gene was selected because there is higher variability in this region among species of the Stenotrophomonas genus in comparison with the 16S rRNA gene.[50] The amplifications were performed with an initial denaturation step at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 68 °C for 45 s and extension at 72 °C for 45 s, with a final extension at 72 °C for 10 min. Stenotrophomonas maltophilia ATCC 13637 was used as a positive control in the assay. All PCR reactions were performed in 25 µL volumes that constituted 50 ng/μL of the template DNA, 0.4 µM of each primer set, 1X Maxima Hotstart PCR master mix (Thermofisher, USA) and nuclease free water.