Biofilme sind eine der widerstandsfähigsten Formen mikrobiellen Lebens. Ihr frühzeitiges Auftreten in der Erdgeschichte konnte durch Stromatolithfunde bewiesen werden. Heutige Biofilme und mikrobielle Matten bieten somit eine Möglichkeit wichtige Einblicke und Erkenntnisse über das erste Leben auf unserem Planeten zu geben. In dieser Arbeit wurden die prokaryotischen Lebensgemeinschaften von verschiedenen Ökosystemen mittels metagenomischer und metatranskriptomischer Methoden analysiert. Mithilfe von „Next-Generation Sequencing“ wurden 16S rRNA Genanalysen, metatranskriptomische Analysen und funktionsbasierte Durchmusterungen von Fosmid-Metagenombanken durchgeführt. Die bakterielle Zusammensetzung und Diversität von kalzifizierenden Biofilmen und dem unterliegenden Kalktuff des Frischwasserbachs Westerhöfer Bach wurden analysiert. Es konnte gezeigt werden, dass der Biofilm hauptsächlich von filamentösen Cyanobacteria, aeroben Vertretern aus allen Klassen der Proteobacteria und Chloroflexi bevölkert wurde. Die bakterielle Diversität nahm flussabwärts zu, was auf Änderungen der physikochemischen Parameter zurückgeführt wird. Aufgrund geringerer UV-Einstrahlung waren im Kalktuff mehr Proteobacteria als Cyanobacteria vorhanden. Des Weiteren gab es deutliche Unterschiede zwischen den relativen Abundanzen der gesamten und aktiven proteobakteriellen Klassen im Biofilm. Die aktiven Funktionen der Biofilm-Mikrobiota einer Westerhöfer Bach Probe wurden mittels metatranskriptomischer Methoden genauer analysiert. Die meisten Transkripte der mikrobiellen Biofilmgemeinschaft umfassten Gene der Photosynthese, des Proteinmetabolismus, des Kohlenstoffmetabolismus und der Zellatmung. Um das metagenomische Potential des Westerhöfer Bach Biofilms zu erschließen, wurden vier „large-insert“ Metagenombanken konstruiert. Funktionsbasierende Durchmusterungsverfahren führten zur Identifikation von fünf bisher unbekannten Genen, die für proteolytische Enzyme kodieren und einem Gen-Cluster, welches für cellulolytische Enzyme kodiert. Bei dem zweiten untersuchten Habitat handelt es sich um eine mikrobielle Matte des hypersalinen Lake 21 auf Kiritimati. Die Mikrobialith-bildende Matte besteht aus neun klar abgegrenzten, unterschiedlich gefärbten Lagen, welche separat auf ihre bakterielle und archaelle Zusammensetzung analysiert wurden. Anhand der prokaryotischen Zusammensetzung und dem Sauerstoff- und Lichtgradienten ergab sich eine Einteilung der mikrobiellen Matte in drei Zonen. Im Allgemeinen erhöhte sich die prokaryotische Diversität mit Tiefe der Matte, wohingegen das Redoxpotential und der pH-Wert sanken. Passend zu den hydrochemischen Daten änderte sich die prokaryotische Zusammensetzung von der photisch-oxischen Zone, welche aus halophilen, oxygenen und anoxygenen Phototrophen und aeroben Heterotrophen bestand, zu Sulfat-reduzierenden Bakterien (SRB), Fermentierern und potentiell Sulfat-reduzierenden Archaeen in der Übergangszone. In der anoxischen Zone konnten hauptsächlich SRB, Fermentierer, Ammonium-oxidierende Archaea und geringe Mengen methanogene Archaeen detektiert werden. Von den kenianischen Natronseen Bogoria, Sonachi, Elementeita und Magadi wurde die prokaryotische Zusammensetzung und Diversität von Boden-, Sediment-, Wasser-, und mikrobiellen Mattenproben analysiert. Hier zeigte sich, dass Boden- sowie Sedimentproben hauptsächlich von Proteobacteria, Gemmatimonadetes, Firmicutes, Actinobacteria, Acidobacteria und Bacteroidetes bevölkert wurden, wohingegen in den Wasserproben Cyanobacteria vorherrschten. Die Archaeen wurden überwiegend von unterschiedlichen Vertretern der Halobacteria repräsentiert. In den humiden Proben wurden außerdem methanogene Archaeen und Thaumarchaeota nachgewiesen. Letztlich wurde in dieser Arbeit die bakterielle Zusammensetzung des Biofilms und des dazugehörigen Planktons von mikrobiellen Brennstoffzellen (MBZ) untersucht. Der erzeugte Datensatz demonstrierte, dass die aktive und gesamte bakterielle Lebensgemeinschaft in den einzelnen Replikaten minimal variierte. Generell zeigte sich, dass stromproduzierende MBZ eine niedrigere bakterielle Diversität aufwiesen als nicht stromproduzierende MBZ. Des Weiteren zeigte die Analyse, dass bisher unkultivierte Vertreter der Spezies Geobacter und Clostridium mit der Stromproduktion verbunden waren.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

O sistema de expressão em Escherichia coli foi o primeiro a ser utilizado para produzir produtos farmacêuticos recombinantes e tem muitas vantagens quando comparado com sistemas eucarióticos, como o fácil cultivo, baixo custo e alto potencial de produção. O fator estimulador de colônias de granulócito (G-CSF) atua principalmente promovendo a maturação dos neutrófilos e estimulando sua atividade fagocítica e quimiotática, além de estar envolvido com o processo de segmentação nuclear dessas células. O fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) tem sido produzido por engenharia genética em Escherichia coli, e é usado no tratamento de diversas patologias, sobretudo em neutropenias provocadas pela quimioterapia usada no tratamento de tumores, pela radioterapia e pelo uso de drogas que suprimem a produção de células mieloides. Desse modo, o presente estudo teve como objetivo a expressão da proteína rhGCSF em bactérias Escherichia coli. A clonagem do gene rhG-CSF no vetor de expressão pET-28a(+) foi realizada nos sítios de restrição das enzimas EcoRI e XhoI, e a expressão da proteína recombinante em cepas de bactéria Escherichia coli BL21DE3 foi obtida com sucesso. A proteína rhG-CSF, fundida à cauda de seis histidinas, foi purificada com êxito e identificada pelas técnicas de Western Blotting e por espectrometria de massas. São necessários estudos para avaliar a integridade estrutural e atividade biológica da proteína produzida, que se confirmada, possibilita que esta seja produzida em escala piloto. / The expression system in Escherichia coli was the first to be used to produce recombinant pharmaceuticals and has many advantages compared to eukaryotic systems, such as easy cultivation and high production potential at low costs. The granulocyte colony (G-CSF) stimulating factor acts primarily by promoting the maturation of neutrophils and stimulating their phagocytic and chemotactic activity. G-CSF is also involved with the process of neutrophils nuclear segmentation. The recombinant human granulocyte colonies stimulating factor (rhG-CSF) has been produced by genetic engineering in Escherichia coli, and it is used to treat of several conditions, especially neutropenia caused by chemotherapy used in the treatment of tumors, by radiotherapy and by the use of drugs that suppress the production of myeloid cells. The present study aimed the expression of rhG-CSF protein in Escherichia coli bacteria. The cloning of rhG-CSF gene in the expression vector pET- 28a (+) was carried out on the restriction sites of the EcoRI and XhoI enzymes. Expression of the recombinant protein in Escherichia coli BL21DE3 was successfully achieved. The rhG-CSF protein, fused with a six histidine tag, was obtained and successfully purified and identified by the Western Blotting and by mass spectrometry techniques. Studies are needed to assess the structural integrity and biological activity of the protein produced, which, if confirmed, enables the production on a pilot scale.

The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis. We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression. We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.

No description available.

Copy number variation detection in prokaryotic organisms is currently receiving more and more attention, mainly due to the association of CNV with pathogenicity and antibiotic resistance in bacteria. The algorithm designed in this thesis uses peak detection in sequencing coverage to detect CNV segments. Read coverage is commonly obtained by mapping sequencing reads of one individual to an already known reference of another individual of the same species. However, two individuals will always differ in a certain number of genes, resulting in unmapped reads that are unnecessarily discarded. Therefore, this work assumes that the biological accuracy of CNV detection can be increased by using a new reference that is created from the same set of reads as the reads mapped to this reference. Sequencing reads of Klebsiella pneumoniae individuals are used to verify this assertion.

Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...

Philosophiae Doctor - PhD / This chapter outlines basic gene structure and how gene structure is related to promoter structure in both prokaryotes and eukaryotes and their transcription machinery. An in-depth discussion is given on variations types of the promoters among both prokaryotes and eukaryotes and as well as among three prokaryotic organisms namely, E.coli, B.subtilis and Mycobacteria with emphasis on Mituberculosis. The simplest definition that can be given for a promoter is: It is a segment of Deoxyribonucleic Acid (DNA) sequence located upstream of the 5' end of the gene where the RNA Polymerase enzyme binds prior to transcription (synthesis of RNA chain representative of one strand of the duplex DNA). However, promoters are more complex than defined above. For example, not all sequences upstream of genes can function as promoters even though they may have features similar to some known promoters (from section 1.2). Promoters are therefore specific sections of DNA sequences that are also recognized by specific proteins and therefore differ from other sections of DNA sequences that are transcribed or translated. The information for directing RNA polymerase to the promoter has to be in section of DNA sequence defining the promoter region. Transcription in prokaryotes is initiated when the enzyme RNA polymerase forms a complex with sigma factors at the promoter site. Before transcription, RNA polymerase must form a tight complex with the sigma/transcription factor(s) (figure 1.1). The 'tight complex' is then converted into an 'open complex' by melting of a short region of DNA within the sequence involved in the complex formation. The final step in transcription initiation involves joining of first two nucleotides in a phosphodiester linkage (nascent RNA) followed by the release of sigma/transcription factors. RNA polymerase then continues with the transcription by making a transition from initiation to elongation of the nascent transcript.