Translational Repression of Bacteriophage T4 DNA Polymerase Biosynthesis

The research described in this dissertation elucidated the mechanism by which bacteriophage T4 DNA polymerase regulates its own biosynthesis. Utilizing both in vivo and in vitro studies, I have shown that autogenous repression occurs at the level of translation. While T4 mutants defective in the structural gene for DNA polymerase (gene 43) overproduce the protein product (gp43) in vivo, they do not overproduce the corresponding mRNA. In vitro, purified DNA polymerase specifically inhibited the translation of its own transcripts. Further, it was demonstrated that gp43 binds its own mRNA at a site overlapping the ribosome initiation domain. Thus, T4 DNA polymerase is a specific translational repressor that presumably inhibits initiation of translation. The mRNA binding site (translational operator) for DNA polymerase includes 38-40 nucleotides upstream of the initiator AUG. The 5\u27 half of this translational operator contains a putative five base-pair stem and 8-base loop, whose existence is inferred from RNase digestion experiments and computer-assisted analysis of RNA folding. To ascertain the important RNA sequence and structural determinants for DNA polymerase binding, I carried out a mutational analysis of the translational operator via the in vitro construction of several operator variants. Operator mutants were subsequently assayed for the effect of each mutation on: 1) gp43/mRNA binding, in vitro 2) the in vivo levels of gp43 biosynthesis from plasmid encoded constructs and 3) in vivo level of gp43 synthesis in phage infections (carried out after introducing mutant operators into the phage genome by virus-plasmid recombination). Mutations that either disrupted the stem or altered particular loop residues, led to diminished binding of purified T4 DNA polymerase in vitro and to derepression of protein synthesis in vivo. Compensatory mutations that restored the stern pairing, with a sequence other than wild-type, restored in vitro binding but still exhibited a mutant phenotype in vivo. Results from loop substitutions suggest that the spatial arrangement of specific loop residues is a major criterion for specific binding of DNA polymerase to its mRNA operator. These studies demonstrate the effectiveness of genetic approaches in dissecting the rules that govern RNA-protein interactions

Cooperativity of Glucocorticoid Response Elements Located Far Upstream of the Tyrosine Aminotransferase Gene

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.Work supported by Grants CSD2008/00013 (to ME and WTC), and BIO2015-69085-REDC and BIO2013-49148-C2-2-R (to ME) from the Spanish Ministry of Economy and Competitiveness; PRPUM grant CG011-2014 (to CCY) from the Malaysian Ministry of Higher Education.Peer reviewedPeer Reviewe

High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (OR3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (OR3) upstream of the prophage repressor gene. The OR3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Funktionelle Untersuchung von genetischen Faktoren mit prognostischer Relevanz in der Onkologie

Lungenkarzinome sind im Vergleich zu anderen Krebsarten die häufigste Todesursache weltweit. Aufgrund der späten Diagnose und einer hohen Rate an Therapieresistenzen liegt die 5-Jahres-Überlebensraten bei nur 13% und ist damit eine der niedrigsten überhaupt. Die Identifikation von potentiellen Biomarkern für eine Verbesserung der Therapieansätze rückt dabei zunehmend in den Vordergrund. Epigenetische Veränderungen spielen bei der Progression von Tumoren eine elementare Rolle, wobei diese mit der Karzinogenese und der Ausprägung einer Therapiereistenz in Verbindung stehen. Epigenetische Modifikatoren wie die H3K4-Methyltransferase KMT2D und das Chromatin-Bindeprotein (Reader) BRD4 sind häufig in verschiedenen Krebsarten wie z.B. in NSCLC mutiert. Im Rahmen dieser Doktorarbeit sollten in verschiedenen Lungenkrebszelllinien unter Verwendung von CRISPR CAS9 definierte Mutationen in den Kandidatengenen KMT2D und BRD4 induziert werden, um funktionelle Mechanismen eingehender zu untersuchen, die mit einer Tumorprogression assoziiert werden. Die Deletionen im Kandidatengen KMT2D unterscheiden sich dadurch, dass bei einer der Deletionen ein Verlust der SET-Domäne erfolgt (Zelllinie B6), wohingegen bei der anderen Deletion das Leseraster C-terminal und damit auch die SET-Domäne erhalten bleibt (Zelllinie A76). In BRD4 wird eine Deletion des gesamten Gens eingefügt. In ChIP-Seq –Analysen der Parental-Zelllinien erfolgte zunächst die Identifikation spezifisch angereicherter Signalwege, die in Prozessen der zellulären Stressantwort, dem Spleißprozess und der epigenetischen Regulation eine Rolle spielten. Zudem konnte eine starke Anreicherung im EGFR-Signalweg festgestellt werden. Um die mögliche Auswirkung der induzierten Mutationen in diesen identifizierten Signalwegen eingehender zu betrachten, wurden diese in Expressions-, Proliferations- und methylierungsspezifischen Experimenten analysiert. Des Weiteren wurde der Einfluss von KMT2D auf eine epigenetisch bedingte Therapieresistenz gegen die EGFR-wirkenden Chemotherapeutika Cetuximab und Erlotinib untersucht. Die induzierten Mutationen führten zu einer verminderten RNA- und Proteinexpression der Kandidatengene. Bei der KMT2D-Zelllinie A76 konnten deutliche morphologische Veränderungen beobachtet werden. Die Zellproliferation war in der Analyse verglichen zur Parental-Zelllinie HCC827 deutlich in den KMT2D-mutierten Zelllinien vermindert. In den Western Blot Analysen führten die induzierten KMT2D- und BRD4-Mutationen zu Veränderung der gesamten Histon-Mengen, die sich in einer differenziellen H3K4- und H3K27-Methylierung in den mutierten Zelllinien widerspiegelten. Durch einen Hitzestress konnte eine Stabilisierung der methylierten Histon-Mengen beobachtet werden. Zudem wurden Unterschiede in der Zellviabilität nach der Induktion durch einen Hitzestress ermittelt. In den BRD4-mutierten Zelllinien konnten hingegen keine Unterschiede in der Zellviabilität nach einem Hitzeschock im Vergleich zur parentalen Zelllinie Wi38 festgestellt werden. Die KMT2D-Mutationen resultierten in einer veränderten RNA-Expression von H3K4- und H3K27-relevanter Methyltransferasen und Demethylasen. Zudem konnten Veränderungen in der Expressionsanalyse von U2 snRNP Spleiß-Faktoren in den KMT2D- und BRD4-mutierten Zelllinien festgestellt werden. Bei der KMT2D-Mutante A76 konnte zudem in Zellviabilitätsassays eine erhöhte Sensitivität gegenüber Cetuximab und Erlotinib beobachtet werden

Regulation of the hrp Type III Secretion System in Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringae pv. DC3000 is a gram-negative bacterium that infects the model plant Arabidopsis thaliana. Pathogenicity is achieved via secretion of effector proteins into the host cytoplasm through a Type III Secretion System (T3SS). In Ps. DC3000 the T3SS (and associated effector proteins) are dependent on HrpL for their transcription. hrpL transcription is sigma54-dependent and requires two co-dependent enhancer binding proteins, HrpR and HrpS (HrpRS), for activation. HrpRS are regulated by two hrpL-dependent proteins, HrpV and HrpG, where HrpV negatively affects HrpRS activity and HrpG relieves this repression. Here the mechanism of HrpV and HrpG’s action on HrpRS activity was tested in vivo and in vitro; and the molecular determinants of HrpV and HrpG functionality were characterised by in silico and mutational analysis. Whole-gene deletion mutants of hrpV and hrpG in Ps. DC3000 revealed complications associated with inserting marker cassettes in transcriptionally-antagonistic orientations. Truncation mutants of HrpV and HrpG showed that C-terminal helices in both proteins play a functional and/or structural role. Alanine mutants indicated a structural role for residues 108-110 in HrpV and a functional role for residues 111-113 in HrpG (NQR motif). HrpV was demonstrated to form a dimer and represses the activity of HrpRS (in vivo and in vitro) but not via inhibiting ATPase activity or destabilising open promoter complexes. HrpG was shown to destabilise the HrpV dimer (potentially via the C-terminal NQR motif). Findings from this study were used to propose a revised model of hrpL regulation in which an active dimer of HrpV binds two adjacent HrpS subunits of a HrpRS hexamer to “lock” the hexamer in an unfavourable conformation. HrpG acts by destabilising the HrpV dimer into an inactive monomeric form. Additional experiments to confirm this model include creating an artificially linked dimer of HrpV and testing its repressive action on HrpRS in the presence or absence of HrpG