核糖体开关的研究进展

T7 RNA polymerase-catalyzed in vitro run-off transcription system
The RNA to have a purine rich sequence at the 5' terminus.
The subsequent RNA is heterogenous in length due to run-off transcription, T7 RNA polymerase tends to add extra nucleotides to the 3-end of the desired RNA.
Work report
Jingen Xu
contents
Methods for the Large-scale Preparation of RNA Suitable for Crystallographic Studies
Work progress for LGR5 and NASP proteins
The hammerhead ribozyme (HH) and VS ribozyme substrate stem–loop (VSsl) are in lower case. Bonds that are cleaved by the ribozymes are shown in broken lines.
Production of homogeneous d56 RNA with use of cis- and trans-ribozymes
Lane A, d56; lane B, d56-d; lane C, d56-VSsl in the absence of VS ribozyme; lane D, as C with VS ribozyme added; lane E, HH-d56-VSsl without VS ribozyme; lane F, same as E, with VS added. Transcription reactions (10 ml each) contained 50 mg/ml linearized plasmid DNA
Turnover cycle of RNA cleavage by a hammerhead ribozyme
Both of turnover and specificity are affected by binding arm length (helix I and III). If the length of binding arms is very short, the rate of dissociation of the target from the ribozyme may exceed the rate of cleavage, resulting in poor efficiency (Rossi, 1997). However, stable hybrids exhibit low catalytic activity because of slow dissociation of the cleaved substrate (Bertrand, 1994). Thus, the ideal situation is to have arm lengths that aid cleavage, yet provide for quick dissociation of the cleaved products.
Note that the first 11 nt of domain V are complementary to a portion of HH
Βιβλιοθήκη Baidu
Nucleic Acids Research, 1996, Vol. 24, No. 5 977–978
Varkud satellite (VS) ribozyme
2.Both of turnover and specificity are affected by binding arm length (helix I and III). If the length of binding arms is very short, the rate of dissociation of the target from the ribozyme may exceed the rate of cleavage, resulting in poor efficiency (Rossi, 1997). However, stable hybrids exhibit low catalytic activity because of slow dissociation of the cleaved substrate (Bertrand, 1994). Thus, the ideal situation is to have arm lengths that aid cleavage, yet provide for quick dissociation of the cleaved products. 3.Divalent cations are believed to participate in the cleavage reaction with Mg 2+ and Mn ~ being preferred
Furin domains confer the relative activity of RSpo proteins
R-spondins function as ligands of the orphan receptors LGR4 and LGR5
Whole-Cell Binding Analysis
it is predicted that HSPGs affect cell surface binding of RSPO, the addition of soluble heparin (a sugar moiety similar to the one found on HSPGs) or sodium chlorate (an inhibitor of sulfatases mediating sulfation of HSPGs) to the medium during culture of cells overexpressing Rspos significantly enhanced the detection of secreted RSPO proteins in the conditioned medium Furthermore, deletion of the TSR domain from RSPOs resulted in an increase of soluble RSPO proteins in conditioned medium
RNA (2004), 10:988–995.
5’AGCCGGCCATGGTCCCAGCCTCCTCGCTGGCGGCCGGTGG GCAACATCGTTCGCGATGGCGAATGGGACC
Work progress
LGR5
NASP and histones
Adult Stem Cell-Driven Epithelial Renewal in the Small Intestine
Ribozyme
Ribozymes are antisense RNA molecules that have catalytic activity. They function by binding to the target RNA moiety through Watson-Crick base pairing and inactivate it by cleaving the phosphodiester backbone at a specific cutting site. They may catalyze self-cleavage (intramolecular or "in-cis" catalysis) as well as the cleavage of external substrates (intermolecular or "in-trans" catalysis)
However, sometimes the well-characterized hammerhead has sequence requirements 5’ to the cleavage site that are incompatible with desired product.
Varkud satellite (VS) ribozyme
Use of cis- and trans-ribozymes to remove 5’ and 3’ heterogeneities from milligrams of in vitro transcribed RNA
1.sequence requirements for cleavage immediately upstream of the cleavage site may be approximated to a NUX (N = rA, rC, rG or rU, X = rA, rC or rU) trinucleotide.
Hammerhead ribozyme Hepatitis delta virus ribozyme Varkud satellite (VS) ribozyme Hairpin ribozyme
The hairpin ribozyme
The name of hammerhead ribozyme is given by the similarity between its secondary structure and the shape of a hammerhead The hammerhead ribozyme is an antisense RNA. Some of the ribonucleotides within the sequence selectively form Watson-Crick base pairs with others to form a stem, while the rest stay in single stranded state called loop.
Hepatitis delta virus ribozyme
The hepatitis delta virus (HDV) ribozyme is a non-coding RNA that is necessary for viral replication and is thought to be the only catalytic RNA known to be required for viability of a human pathogen. The ribozyme acts to process the RNA transcripts to unit lengths in a self-cleavage reaction
The N-terminal domains of LGR5 are required for RSPO1 binding
Wnt signaling pathway
Schematic representation of four human RSPO proteins
HSPG:heparin sulfate proteoglycans肝素蛋白多糖
Cristin/R-spondins are secreted proteins