Академический Документы
Профессиональный Документы
Культура Документы
Housekeeping ncRNAs
tRNA and rRNA - translation snRNA Pre-mRNA splicing snoRNA rRNA modification gRNA guide RNA in RNA editing Telomerase RNA primer for telomeric DNA synhesis A few other...
SRP
ribosome
mRNA
Structure of SRP
RNA holds together the protein subunits of SRP as well as helps to bind to ribosome
If the termination codon by some mistake is not reached, the ribosome gets stack upon the reaching of 3 end of mRNA and has to be rescued Since the stop codon is not reached, the newly made protein is probably wrong and needs to be degraded
Ala
The tRNA part of tmRNA (black) adds an alanine to the growing polypeptide chain The mRNA part (red) enters the ribosome and the synthesis of polypeptide is continued with aid of normal tRNAs (blue), until the termination codon is reached In the end, ribosome is released and the newly made fusion protein is degraded due to the signal sequence in Cterminus
Regulatory ncRNAs
Transcriptional regulators Translational regulators Modulators of protein function Regulators of RNA and protein distribution
Dosage compensation
In animals, males and females have different number of X chromosomes (e.g. 1 or 2) To equalize the expression levels from X chromosome in males and females some sort of mechanism must exist, called dosage compensation
MSL-2
MSL-1
MSL complex has about 35 entry sites in Drosophila genome. Two of them actually contain roX1/rox2 gene. This suggests a possible role of rox1/rox2 RNAs in entry site recognition
Acetylated lysines
Translational regulation
Translational regulation by ncRNAs is achieved by anti-sense mechanism, when ncRNA binds to target mRNA RNA interference covered separately in the end of this lecture
Ribosome
Anti-sense RNA
DsrA RNA in E.coli activates ribosome binding to stress-response s factor rpoS mRNA
DsrA RNA Ribosome binding site blocked by base-pairing
rpoS mRNA
RBS accessible
s70
RNA pol
6S RNA
Ribozymes
RNA molecules with catalytical properties (Ribonucleic acid enzymes) In nature ribozymes occur mostly within self-splicing intrones and RNA encoded parasites satellites and viroids The catalyzed reactions in naturally occuring ribozymes are limited to cleavage and ligation of RNA Some researchers consider even ribosomes being ribozymes, since the peptide bond formation is catalyzed by RNA Most naturally occuring ribozymes act on themselves The catalytical efficiency of ribozymes is typically much lower (~1000-fold) than of analogous protein enzymes Several synthetic ribozymes are cabaple of performing other reactions than RNA cleavage and ligation
Cleaving ribozymes
Cleavage
Other classes of cleaving ribozymes do not require metal ion for activity. Amino group of nearby nucleotide base destabilizes the phosphodiester bond instead
Cyt
Ligating ribozymes
Reaction mechanism similar to that of RNA polymerases, requires Mg ion for catalysis
RNAse P
RNAse P is a ribozyme
RNAse P cleaves the 5 end of pre-tRNAs It is composed of 12 kDa P protein and about 400 nt long RNA The catalytic activity lies entirely within RNA part Enzyme is efficient without P protein but in high salt conditions P protein or high salt is thought to screen the repulsive electrostatic interactions between RNAse P RNA and substrate pre-tRNA
Revolutionary new technology (potent and simple) to knock down gene expression in eukaryotic cells
year
The injection of double-stranded RNAs into C. elegans resulted in the silencing of a gene complementary to dsRNAs.
A- negative control (without hybridization probe) B- normal pattern of endogenous mex-3 RNA C- injected with antisense RNA D- injected with dsRNA
RNAi in
Fungi, plants and worms
systemic nature of
non heritable
no indication of siRNA replication
miRNAs do not induce target cleavage but block translation by binding to complementary mRNAs
miRNAs are encoded by the host genome, whereas siRNAs in most cases originate from outer source
Isolated from a pool of about 1015 synthetic RNAs, based on ligating ribozyme Fidelity of 96.7 % Extension time: 14 nucleotides in 24 hours