A systems approach to model natural variation in reactive properties of bacterial ribosomes

<p>Abstract</p> <p>Background</p> <p>Natural variation in protein output from translation in bacteria and archaea may be an organism-specific property of the ribosome. This paper adopts a systems approach to model the protein output as a measure of specific ribosome reactive properties in a ribosome-mediated translation apparatus. We use the steady-state assumption to define a transition state complex for the ribosome, coupled with mRNA, tRNA, amino acids and reaction factors, as a subsystem that allows a focus on the completed translational output as a measure of specific properties of the ribosome.</p> <p>Results</p> <p>In analogy to the steady-state reaction of an enzyme complex, we propose a steady-state translation complex for mRNA from any gene, and derive a maximum specific translation activity, <it>T</it>_{<it>a</it>(max)}, as a property of the ribosomal reaction complex. <it>T</it>_{<it>a</it>(max)}has units of <it>a</it>-protein output per time per <it>a</it>-specific mRNA. A related property of the ribosome, <inline-formula><m:math name="1752-0509-2-62-i1" xmlns:m="http://www.w3.org/1998/Math/MathML"><m:semantics><m:mrow><m:msub><m:mover accent="true"><m:mi>T</m:mi><m:mo>˜</m:mo></m:mover><m:mrow><m:mi>a</m:mi><m:mo stretchy="false">(</m:mo><m:mi>max</m:mi><m:mo>⁡</m:mo><m:mo stretchy="false">)</m:mo></m:mrow></m:msub></m:mrow><m:annotation encoding="MathType-MTEF"> MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmivaqLbaGaadaWgaaWcbaGaemyyaeMaeiikaGIagiyBa0MaeiyyaeMaeiiEaGNaeiykaKcabeaaaaa@3464@</m:annotation></m:semantics></m:math></inline-formula>, has units of <it>a</it>-protein per time per total RNA with the relationship <inline-formula><m:math name="1752-0509-2-62-i1" xmlns:m="http://www.w3.org/1998/Math/MathML"><m:semantics><m:mrow><m:msub><m:mover accent="true"><m:mi>T</m:mi><m:mo>˜</m:mo></m:mover><m:mrow><m:mi>a</m:mi><m:mo stretchy="false">(</m:mo><m:mi>max</m:mi><m:mo>⁡</m:mo><m:mo stretchy="false">)</m:mo></m:mrow></m:msub></m:mrow><m:annotation encoding="MathType-MTEF"> MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmivaqLbaGaadaWgaaWcbaGaemyyaeMaeiikaGIagiyBa0MaeiyyaeMaeiiEaGNaeiykaKcabeaaaaa@3464@</m:annotation></m:semantics></m:math></inline-formula> = <it>ρ</it>_{<it>a </it>}<it>T</it>_{<it>a</it>(max)}, where <it>ρ</it>_{<it>a </it>}represents the fraction of total RNA committed to translation output of <it>P</it>_{<it>a </it>}from gene <it>a </it>message. <it>T</it>_{<it>a</it>(max)}as a ribosome property is analogous to <it>k</it>_catfor a purified enzyme, and <inline-formula><m:math name="1752-0509-2-62-i1" xmlns:m="http://www.w3.org/1998/Math/MathML"><m:semantics><m:mrow><m:msub><m:mover accent="true"><m:mi>T</m:mi><m:mo>˜</m:mo></m:mover><m:mrow><m:mi>a</m:mi><m:mo stretchy="false">(</m:mo><m:mi>max</m:mi><m:mo>⁡</m:mo><m:mo stretchy="false">)</m:mo></m:mrow></m:msub></m:mrow><m:annotation encoding="MathType-MTEF"> MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmivaqLbaGaadaWgaaWcbaGaemyyaeMaeiikaGIagiyBa0MaeiyyaeMaeiiEaGNaeiykaKcabeaaaaa@3464@</m:annotation></m:semantics></m:math></inline-formula> is analogous to enzyme specific activity in a crude extract.</p> <p>Conclusion</p> <p>Analogy to an enzyme reaction complex led us to a ribosome reaction model for measuring specific translation activity of a bacterial ribosome. We propose to use this model to design experimental tests of our hypothesis that specific translation activity is a ribosomal property that is subject to natural variation and natural selection much like <it>V</it>_maxand <it>K</it>_mfor any specific enzyme.</p

Regulation-Structured Dynamic Metabolic Model Provides a Potential Mechanism for Delayed Enzyme Response in Denitrification Process

In a recent study of denitrification dynamics in hyporheic zone sediments, we observed a significant time lag (up to several days) in enzymatic response to the changes in substrate concentration. To explore an underlying mechanism and understand the interactive dynamics between enzymes and nutrients, we developed a trait-based model that associates a community’s traits with functional enzymes, instead of typically used species guilds (or functional guilds). This enzyme-based formulation allows to collectively describe biogeochemical functions of microbial communities without directly parameterizing the dynamics of species guilds, therefore being scalable to complex communities. As a key component of modeling, we accounted for microbial regulation occurring through transcriptional and translational processes, the dynamics of which was parameterized based on the temporal profiles of enzyme concentrations measured using a new signature peptide-based method. The simulation results using the resulting model showed several days of a time lag in enzymatic responses as observed in experiments. Further, the model showed that the delayed enzymatic reactions could be primarily controlled by transcriptional responses and that the dynamics of transcripts and enzymes are closely correlated. The developed model can serve as a useful tool for predicting biogeochemical processes in natural environments, either independently or through integration with hydrologic flow simulators

Ribosoomide heterogeensus bakterites Escherichia coli bL31 paraloogide näitel

Väitekirja elektrooniline versioon ei sisalda publikatsiooneSelleks, et ellu jääda, kasvada ja paljuneda, vajavad organismid sadu erinevaid valke, mis toimivad struktuursete komponentide, ensüümide, signaalivahendajate, transpordi- ja säilitusmolekulidena. Lisaks sellele on elutähtis, et valgud oleksid funktsionaalsed sobivas koguses, õigel ajal ja vajalikus kohas – seetõttu on valgusüntees ja selle regulatsioon kesksemaid eluprotsesse. Kõiki valke sünteesivad ribosoomid, RNA-st ja valkudest koosnevad kompleksid. Bakteri ribosoom, selle doktoritöö uurimisobjekt, koosneb kolmest ribosoomi RNAst ja rohkem kui 50 ribosoomi valgust, mis jagunevad kahe subühiku vahel. Eksperimentaalselt on kindlaks tehtud, et nii päris- kui eeltuumsed organismid sisaldavad mõnevõrra erineva ülesehitusega ribosoome. Samas ei ole selle nähtuse – ribosoomide heterogeensuse – bioloogiline tähtsus teada. Käesoleva doktoritöö fookuses on soolekepikese (E. coli) teatud tüüpi ribosoomi valgud (paraloogid), millel on ühine eellane, kuid mis kodeerivad erinevaid valke. Küsimus on, kas E. coli ribosoomid on paraloogide poolest heterogeensed. Mis võiks olla sellise molekulaarse mitmekesisuse roll valgusünteesil ja bakterite kasvu jaoks? E. coli ribosoomide valgulise koostise analüüs tuvastas, et nii kiire kasvu korral kui statsionaarses kasvufaasis esinevad samaaegselt ribosoomi valkude paraloogide poolest heterogeensed ribosoomid. Kasvukatsed näitasid, et ribosoomi valk bL31 paraloogid (bL31A ja bL31B) on olulised, ent mitte samaväärsed bakterite kasvuks madalamatel temperatuuridel. Nimelt annab bL31A olemasolu bakterirakkudele kiire kasvu faasis kasvueelise võrreldes bL31B-ga. bL31A ja bL31B osalevad üksteisega samaväärselt optimaalse translatsiooni initsiatsiooni etapi kiiruse ja ribosoomi subühikute ühendamise tagamisel. Samas näitavad meie tulemused, et võrreldes bL31B-d sisaldavate ribosoomidega on bL31A-d sisaldavad ribosoomid protsessiivsemad ja teevad vähem vigu valgusünteesi käigus. Doktoritöö tulemused avardavad oluliselt teadmisi ribosoomide heterogeensusest bakterites ning ribosoomi valgu bL31 tähtsusest valgusünteesil.To survive, grow and reproduce all organisms need hundreds of proteins acting as enzymes, messengers, structural components, transport and storage molecules. In addition, proteins are required to be functional at the right place, time and in sufficient amount. Therefore, protein synthesis and its regulation belong to the most central life processes. Proteins are synthesized by RNA-protein complexes called ribosomes. Experimental evidence indicates that eukaryotic and procaryotic organisms produce ribosomes with slightly different structure. The biological meaning of the phenomenon – ribosome heterogeneity – is not known. This thesis focuses on bacterial ribosome heterogeneity originating from a certain type of ribosomal proteins (paralogs) in E. coli. Paralogs have a common ancestor gene, but they encode proteins with different amino acid sequence. How does ribosome heterogeneity in ribosomal protein bL31 paralog content affect bacterial growth and translation? Analysis of ribosomal protein content showed that E. coli ribosomes are heterogeneous with respect to paralogs during fast and stationary growth phase. Subsequent work on bL31 paralogs (bL31A and bL31B) demonstrated that they are important but not equivalent for bacterial growth at lower temperatures because bL31A gives growth advantage over bL31B during fast growth. Both bL31 paralogs contribute to similar extent to translation initiation, especially to subunit joining. Interestingly, bL31A containing ribosomes are more processive and they make less errors during translation as compared to ribosomes with bL31B. This indicates that ribosome heterogeneity in bL31 paralog content may regulate translation. This thesis shed light onto functional importance of bacterial ribosome heterogeneity and thus helps us to better understand its biological meaning.https://www.ester.ee/record=b550899

The ten grand challenges of synthetic life

The construction of artificial life is one of the main scientific challenges of the Synthetic Biology era. Advances in DNA synthesis and a better understanding of regulatory processes make the goal of constructing the first artificial cell a realistic possibility. This would be both a fundamental scientific milestone and a starting point of a vast range of applications, from biofuel production to drug design. However, several major issues might hamper the objective of achieving an artificial cell. From the bottom-up to the selection-based strategies, this work encompasses the ten grand challenges synthetic biologists will have to be aware of in order to cope with the task of creating life in the lab

Cell-Free Protein Synthesis

The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists

Escherichia coli Chromosomes in the Crowded Cellular Environment

\In spite of our detailed knowledge of the enzymology of DNA replication and of the topology of gene expression, we do not understand how, on a larger scale, bacterial DNA is organized within cell or nucleoid. Also, in the process of [bacterial DNA] segregation, we hardly know what force(s) move the newly replicated DNA strands faithfully to the prospective daughter cells. --- Conrad L. Woldringh To provide insights and answers in response to these questions, we have designed pressure actuated micro uidic valves based on a PDMS lab-on-a-chip platform. Using this device, we mechanically perturb the main macromolecular structures in Escherichia coli, and monitor the individual phenotypic responses in real time. Meanwhile, we also utilized the mothermachine design and rened it suiting E. coli cells in particular growth conditions. In the mother-machine devices, we apply osmotic shocks to the cells. We nd mechanical perturbations decrease the cytoplasmic cell volume, which in turn causes the compactness of nucleoid to increase and the chromosome-cytoplasm phase separation to be more abrupt; meanwhile, the chromosome-free regions between adjacent separated nucleoids and at cell poles persist. Furthermore, with an ardent perturbation, nucleoids are rarely bisected; nevertheless, mild perturbations often displace nucleoids after a period of deforming them. I also discuss the possibility to observe the effect of the hypothesized co-transcriptional translation transertion and our results which potentially indicate that

Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments

RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored. Three model rRNA hairpins from E. coli ribosome were selected. Two helix 69 (H69) constructs, modified H69 (with pseudouridine) and unmodified H69 (without pseudouridine), and the 790 loop, which has an identical size and nucleotide composition to unmodified H69, were used. Prior to kinetic studies, cisplatin targets on each RNA were determined using RNase T1 mapping combined with MALDI MS, and dimethyl sulfate (DMS) probing. The kinetic studies were carried out under pseudo-first-order conditions and electrostatic properties were evaluated using Brønsted-Debye-Hückel and polyelectrolyte theories. RNase T1 mapping with MALDI MS and dimethyl sulfate (DMS) probing revealed GpG sites as cisplatin targets on RNA. The DMS probing further revealed platination-induced structural changes in RNA. Both the RNA sequence and modified nucleotides showed an impact on platination rates. Kinetic data showed that the platination rate is dependent on cations and the abundance of active cisplatin complexes. Structure, pseudouridylation, availability of active cisplatin species, and cation/Pt+ electrostatic competitions all impact platination of the two H69 RNAs. Probing neomycin-H69 interactions by platination kinetics indicated that structural changes in modified H69 upon aminoglycoside binding could also impact the platination kinetics. Electrostatic models revealed that nucleotide sequence, cations, and H+ ions impact the global RNA electrostatics. The similar global electrostatic properties between the two H69 RNAs indicated that structure-dependent electrostatic changes in modified H69 could be limited to the loop region. In conclusion, this thesis work showed that both intrinsic RNA characteristics such as structure, sequence, and dynamics, as well as bulk solution conditions (e.g. cations and pH), impact cisplatin-RNA interactions. The RNA electrostatic parameters determined in this thesis work illustrated platination kinetics can be used as an informatory tool for probing dynamic RNA microenvironments

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications