scholarly journals Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

2021 ◽  
Author(s):  
◽  
Elsie May Williams
Keyword(s):  
Pet Imaging ◽  
Targeted Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Phase Iii ◽  
Imaging Agents ◽  
Tumour Hypoxia ◽  
Active Site Residues ◽  
Reporter Protein ◽  
E Coli ◽  
Cancerous Cells

<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A.  This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents.  In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity.  Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

scholarly journals Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

2021 ◽  
Author(s):  
◽  
Elsie May Williams
Keyword(s):  
Pet Imaging ◽  
Targeted Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Phase Iii ◽  
Imaging Agents ◽  
Tumour Hypoxia ◽  
Active Site Residues ◽  
Reporter Protein ◽  
E Coli ◽  
Cancerous Cells

<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A.  This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents.  In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity.  Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

scholarly journals The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase

1994 ◽  
Vol 300 (3) ◽  
pp. 765-770 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
E Coli ◽  
Rate Limiting Step ◽  
Rate Limiting

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

Detection of benzene derivatives by recombinant E. coli with Ps promoter and GFP as a reporter protein

2003 ◽  
Vol 15 (3) ◽  
pp. 193-197 ◽  
Author(s):  
Shinya Ikeno ◽  
Chiaki Ogino ◽  
Takeo Ito ◽  
Nobuaki Shimizu
Keyword(s):  
Benzene Derivatives ◽  
Reporter Protein ◽  
E Coli

scholarly journals E. coli F1 -ATPase: Site-directed mutagenesis of the β-subunit

FEBS Letters ◽  
1988 ◽  
Vol 232 (1) ◽  
pp. 111-114 ◽  
Author(s):  
Derek Parsonage ◽  
Susan Wilke-Mounts ◽  
Alan E. Senior
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
E Coli ◽  
F1 Atpase

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

scholarly journals Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3630-3639 ◽  
Author(s):  
David E. Nelson ◽  
Anindya S. Ghosh ◽  
Avery L. Paulson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Outer Face ◽  
Penicillin Binding Protein ◽  
Membrane Anchor ◽  
E Coli ◽  
Membrane Bound ◽  
Carboxy Terminal ◽  
Membrane Anchoring ◽  
Β Sheet

ABSTRACT Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar dd-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other dd-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the dd-carboxypeptidase enzymatic core.

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

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