mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding [preprint]

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

Diarrheagenic pathogens in adults attending a hospital in Singapore.

BACKGROUND: Singapore's diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults' stool samples collected from a large public hospital. METHODS: The stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used. RESULTS: Pathogens were detected in 32% of the samples. The predominant organisms encountered were norovirus genogroup II (11%), Aeromonas spp. (9%) and Campylobacter spp. (5%). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2% of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1% of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected. CONCLUSION: Our preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities

Regulation of Ribosome Structural Dynamics by Antibiotics, Translation Factors and mRNA Secondary Structure

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2017.The structural dynamics of the ribosome underlie the mechanism by which information encoded in messenger RNAs is translated into a polypeptide chain. During protein synthesis, the small and large ribosome subunits rotate relative to each other while the L1 stalk, a mobile domain of large subunit, moves inward/outward relative to the core of the large subunit. Translation factors, antibiotics and structured RNAs are able to regulate or perturb translation by modulating ribosome structural dynamics. Furthermore, translation factors also undergo large structural rearrangements while interacting with the ribosome. Using single-molecule Förster resonance energy transfer (smFRET) to follow the structural rearrangements of the ribosome and translation factors, I have found (a) that the antibiotic, blastidicin S (BlaS), slows down intersubunit rotation, (b) the HIV and dnaX frameshift stimulating stemloops inhibit ribosome translocation by blocking tRNA binding to the A site of the ribosome, and (c) bacterial initiation factor 2 (IF2) positions ribosome subunits in a semi-rotated orientation during the subunit-joining step of translation initiation. Many small molecule antibiotics that target translation perturb ribosome structural dynamics. BlaS is a potent translation inhibitor in both eukaryotes and bacteria; however, the mechanism of BlaS action was not clear. I found that BlaS interacts with the P-site tRNA and inhibits spontaneous intersubunit rotation in bacterial ribosomes. Further studies by our collaborators from UMASS School of Medicine revealed that BlaS targets proteins synthesis via a unique mechanism hampering translation termination. During translation elongation, the ribosome moves along mRNA in a codon-by- codon manner and unwinds mRNA secondary structure. However, specific mRNA stemloops, such as the frameshift stimulating signal (FSS) from the E. coli dnaX gene, induce ribosome stalling and frameshifting. We sought to determine how FSS RNA stemloops stall ribosome translocation despite efficient helicase activity of the ribosome. Surprisingly, we found that the FSSs from HIV and dnaX mRNAs block tRNA binding to the A site of the ribosome, thereby inhibiting translocation mediated by the bacterial ribosome translocase, elongation factor-G. Our future studies will identify properties of mRNA secondary structure that inhibit A-site tRNA binding. Thus, our studies will provide fundamental insights into the regulation of translation elongation by mRNA structure and the mechanism of programmed ribosome frameshifting. We also extended our studies beyond translation elongation to elucidate the molecular mechanism of the subunit-joining step of translation initiation in bacteria. Initiation of protein synthesis is the key regulatory step of translation. In bacteria, translation initiation is controlled by initiation factors (IFs) 1, 2 and 3, which ensure selection of the initiator tRNA and promote joining of the large and small ribosomal subunits. Using smFRET, we find that IF2, a translational GTPase, transiently stabilizes the ribosome in a distinct conformation where the ribosomal subunits are in a semi- rotated orientation and the L1 stalk is in a half-closed position (3). Our results suggest that positioning subunits in a semi-rotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding

Identification of Y chromosomal material in turner syndrome by Fluorescence in Situ Hybridisation (FISH)

Turner syndrome is one of the most common chromosomal abnormalities affecting newborn females. More than half of patients with Turner syndrome have a 45X karyotype. The rest of the patients may have structurally abnormal sex chromosomes or are mosaics with normal or abnormal sex chromosomes. Mosaicism with a second X sex chromosome is not usually of clinical significance. However, Turner syndrome patients having a second Y chromosome or Y chromosomal material are at risk of developing gonadoblastoma later in life. The aim of this study is to compare the results of conventional (karyotyping) and molecular cytogenetics (FISH), and discuss the advantages and limitations in the diagnosis of Turner syndrome. We also aim to compare the degree of mosaicism identified using conventional cytogenetics and FISH techniques. Conventional cytogenetics and FISH analyses were performed on eight peripheral blood samples of patients with Turner syndrome collected between 2004 and 2006. From this study, two out of eight patients with Turner syndrome were found to have the sex determining region on the Y chromosome (SRY) gene by FISH analysis. Our results showed that the rate of detection of mosaic cases in Turner syndrome was also increased to 88% after using the FISH technique. We concluded that FISH is more superior to conventional cytogenetics in the detection of the Y chromosomal material. FISH is also a quick and cost effective method in diagnosing Turner syndrome and assessing the degree of mosaicis

Digital Organism Simulation Environment (DOSE): A Library for Ecologically-Based In Silico Experimental Evolution

Testing evolutionary hypothesis in biological setting is expensive and time consuming. Computer simulations of organisms (digital organisms) are commonly used proxies to study evolutionary processes. A number of digital organism simulators have been developed but are deficient in biological and ecological parallels. In this study, we present DOSE (Digital Organism Simulation Environment), a digital organism simulator with biological and ecological parallels. DOSE consists of a biological hierarchy of genetic sequences, organism, population, and ecosystem. A 3-character instruction set that does not take any operand is used as genetic code for digital organism, which the 3-nucleotide codon structure in naturally occurring DNA. The evolutionary driver is simulated by a genetic algorithm. We demonstrate the utility in examining the effects of migration on heterozygosity, also known as local genetic distance

Resistant traits in digital organisms do not revert preselection status despite extended deselection : implications to microbial antibiotics resistance

Antibiotics resistance is a serious biomedical issue as formally susceptible organisms gain resistance under its selective pressure. There have been contradictory results regarding the prevalence of resistance following withdrawal and disuse of the specific antibiotics. Here, we use experimental evolution in “digital organisms” to examine the rate of gain and loss of resistance under the assumption that there is no fitness cost for maintaining resistance. Our results show that selective pressure is likely to result in maximum resistance with respect to the selective pressure. During deselection as a result of disuse of the specific antibiotics, a large initial loss and prolonged stabilization of resistance are observed, but resistance is not lost to the stage of preselection. This suggests that a pool of partial persists organisms persist long after withdrawal of selective pressure at a relatively constant proportion. Hence, contradictory results regarding the prevalence of resistance following withdrawal and disuse of the specific antibiotics may be a statistical variation about constant proportion. Our results also show that subsequent reintroduction of the same selective pressure results in rapid regain of maximal resistance. Thus, our simulation results suggest that complete elimination of specific antibiotics resistance is unlikely after the disuse of antibiotics once a resistant pool of microorganisms has been established.Published versio

Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome

The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-A crystal structure of BlaS bound to a 70StRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3\u27 terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation

Relationships between writing motives, writing self-efficacy and time on writing among Chinese students : Path models and cluster analyses

Motives for writing is a new research area in the field of writing motivation. Recent studies have identified important motives for students’ writing. Adding to this nascent foundation, the current study examined relationships between writing motives, writing self-efficacy and time spent on writing using a sample of grade 4 Chinese students in Shanghai (n = 619) who completed a questionnaire assessing these constructs. Four alternative models, driven separately by key motives including social recognition, curiosity, competition and grades, depicted complex relationships between motives, self-efficacy and time on writing. The grades driven model best-fitted the data. This final model showed the importance of grades as a key motive for writing and how intrinsic and extrinsic motivational pathways were originated from this performance-focused motive, connecting with writing self-efficacy and time spent on writing. Cluster analyses classified these Chinese students into seven groups of beginning writers holding contrasting profiles of writing motives. Extremely-motivated and highly-motivated writers held multiple motives in their profiles. The motive profiles of curious and averagely-motivated writers were dominated by curiosity and involvement. Performance-focused and weakly-motivated writers focused predominantly on grades as a key motive. Finally, unmotivated writers did not hold any clear motives for writing. These clusters of writers differed in writing self-efficacy but not in their time spent on writing. Complementing the findings of path analyses, cluster analyses showed grades as a dominant motive for writing among different clusters of motivated writers