Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase

Gruegelsiepe, Heike; Schn, Astrid; Kirsebom, Leif A.; Hartmann, Roland K.

doi:10.1002/9783527619504.ch1

Cited by 9 publications

(6 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Active natural glycosylated PAI-1 as well as recombinant PAI-1 was prepared as described previously (Dupont et al 2006). The mutant T7 RNA polymerase Y639F was expressed and prepared essentially as described previously (Grügelsiepe et al 2005). The monoclonal antibody to uPA (antiuPA clone 6) was described previously , as was the rabbit polyclonal anti-uPA antibody (Knoop et al 1998).…”

Section: Proteins and Antibodiesmentioning

confidence: 99%

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

Dupont¹,

Madsen²,

Hartmann³

et al. 2010

RNA

View full text Add to dashboard Cite

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 29-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 29-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

show abstract

Section: Proteins and Antibodiesmentioning

confidence: 99%

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

Dupont¹,

Madsen²,

Hartmann³

et al. 2010

RNA

View full text Add to dashboard Cite

show abstract

“…S30 cell extracts were prepared from the E. coli A19 strain as described in detail elsewhere . T7 RNA polymerase was prepared according to available protocols . Cell‐free protein synthesis was performed in the continuous‐exchange configuration with D‐tube™ dialyzers containing the reaction mix (RM) in different sizes (D‐tube™ mini/midi/maxi; Merck) which were put into 2 mL tubes (mini) or 50 mL tubes (midi/maxi) harboring the feeding mix (FM).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of cell‐free protein synthesis for the crystallization of membrane proteins – a case study on a member of the glutamate transporter family from Staphylothermus marinus

Jaehme

Michel

2013

The FEBS Journal

View full text Add to dashboard Cite

Cell‐free in vitro synthesis of proteins using coupled transcription/translation is considered to be a powerful alternative to the use of traditional cell‐based expression systems. Recently, promising developments have been reported applying cell‐free production to membrane proteins for structural biology and in particular for NMR spectroscopy. However, the general applicability of this system to produce large amounts of stable, functional and homogeneous membrane proteins as required for X‐ray crystallography remains to be determined. Here, we present a systematic study comparing structural and functional properties of membrane proteins produced using Escherichia coli derived in vitro and in vivo expression systems. The function of the target membrane protein, a previously uncharacterized bacterial glutamate transporter homolog from Staphylothermus marinus, was analyzed using ligand binding and transport assays. In addition, the protein structure was investigated with respect to its overall fold and oligomeric state in different detergents. We found that the protein synthesized in vitro is highly stable and monodisperse. However, in contrast to the protein produced using an in vivo system, it was not able to assemble into the native trimeric state nor to bind substrate. We thus conclude that cell‐free expression systems can compromise folding and function of such complex secondary active transporters. The expression product has to be carefully characterized prior to biophysical investigations like crystallography of membrane proteins. Structured digital abstract GltSM and GltSM bind by molecular sieving ( View interaction) GltSM and GltSM bind by cross-linking study ( View interaction)

show abstract

“…In vitro transcription, 32 P-labeling, and structure probing with RNase T1 B. subtilis P RNA variants and pre-tRNA Gly (from T. thermophilus) were produced by runoff in vitro transcription using plasmid pDW66 and mutant constructs thereof (linearized with DraI; Warnecke et al 1999), and pSBpt3 ′ hh (linearized with BamHI; Busch et al 2000), respectively, as previously described (Gruegelsiepe et al 2005). 5 ′ -End-labeling of pre-tRNA and 5 ′and 3 ′ -end-labeling of P RNAs were done as previously described (Gimple and Schön 2014).…”

Section: Inhibition Assaysmentioning

confidence: 99%

Bacterial type B RNase P: functional characterization of the L5.1-L15.1 tertiary contact and antisense inhibition

Walczyk

Willkomm

Hartmann

2016

RNA

Self Cite

View full text Add to dashboard Cite

Ribonuclease P is the ubiquitous endonuclease that generates the mature 5'-ends of precursor tRNAs. In bacteria, the enzyme is composed of a catalytic RNA (∼400 nucleotides) and a small essential protein subunit (∼13 kDa). Most bacterial RNase P RNAs (P RNAs) belong to the architectural type A; type B RNase P RNA is confined to the low-G+C Gram-positive bacteria. Here we demonstrate that the L5.1-L15.1 intradomain contact in the catalytic domain of the prototypic type B RNase P RNA of Bacillus subtilis is crucial for adopting a compact functional conformation: Disruption of the L5.1-L15.1 contact by antisense oligonucleotides or mutation reduced P RNA-alone and holoenzyme activity by one to two orders of magnitude in vitro, largely retarded gel mobility of the RNA and further affected the structure of regions P7/P8/P10.1, P15 and L15.2, and abolished the ability of B. subtilis P RNA to complement a P RNA-deficient Escherichia coli strain. We also provide mutational evidence that an L9-P1 tertiary contact, as found in some Mycoplasma type B RNAs, is not formed in canonical type B RNAs as represented by B. subtilis P RNA. We finally explored the P5.1 and P15 stem-loop structures as targets for LNA-modified antisense oligonucleotides. Oligonucleotides targeting P15, but not those directed against P5.1, were found to efficiently anneal to P RNA and to inhibit activity (IC of ∼2 nM) when incubated with preassembled B. subtilis RNase P holoenzymes.

show abstract

Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase

Cited by 9 publications

References 32 publications

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

Evaluation of cell‐free protein synthesis for the crystallization of membrane proteins – a case study on a member of the glutamate transporter family from Staphylothermus marinus

Bacterial type B RNase P: functional characterization of the L5.1-L15.1 tertiary contact and antisense inhibition

Contact Info

Product

Resources

About