RNA Degradation in Bacteria Flashcards
Was ist die a. doubling time b. average mRNA half-lives von 1.E.coli 2.S.cerivisiae 3.Human
1.a. 20-60min b.3-5min
2.a. 180min b.22min
3.a. 16-24h b.10h
Was sind die Schritte zur RNA Halbwertszeit Messung?
- Kultivierung
- Rifampicin addition
- sampling
- RNA Preparation
- Northern blotting/ qRT-PCR
- Detection
- Reprobing
- Detection
- Normalization
- Calculation
Was ist Riampicin?
polyketide Antibiotika, produziert von Soil Bakterien, die RNAP inhibieren
Was sind Ribonucleasen? RNasen?
- Nukleasen, die RNA zersetzen
- Spaltung von Phosphordiesterbindungen in RNA Substraten
Wie viele Enzymklassen und Subgruppen gibt es?
2 und 2
EC2.7 phosphorolytic enzymes
EC3.1 hydrolytic enzymes
und Subgruppen:
endoribonuclease
exoribonuclease
Welche Funktionen haben RNasen?
mRNA turnover
maturation of stable RNAs degradation of replication primer
sRNA mediated gene regulation
defense against horizontal gene transfer
Was ist die RNA Reifung
Stable RNAs (tRNA, rRNA)
All precursors of stable RNAs undergo maturation steps
tRNA: 5‘ end cleavage by RNAse P
rRNA: endonucleolytic cleavage of 30S
pre-rRNA transcript
Exonucleolytic processing at 3‘ ends
Base modification
Was ist die RNA Degradierung?
All RNAs (mRNAs, sRNAs, stable
RNAs, defective RNAs…)
Combination of endo- and
exoribonucleolytic cleavages Tightly coupled with translation
depends on translation frequency
active translation protects
Wie läuft das rRNA Processing der rRNA Reifung ab?
Division of the polycistronic precursor
Removal of nucleotides from the primary transcript Addition of nucleotides with sequences not encoded by the corresponding genes Covalent modification of certain bases
Wie läuft die tRNA Reifung ab?
tRNAs genes are encoded as:
- single gene
- tRNA Operons
- part of rRNA operon
tRNA processing includes:
Separation of the single precursor tRNAs
endonucleases
Endonucleolytic 5’ end generation
Always RNase P
Exonucleolytic 3’ end trimming
Activity of different exoribonucleases depending on the tRNA gene
Base modification
Was schützt eine mRNA vor den Abbau von Endoribonucleasen?
Triphosphate an einem Ende; Stem Loop Ende
Was sind die Endonucleasen?
I. Internal cleavage by an endonuclease
RNase E (E. coli)
RNase Y (B. subtilis)
RNase II
Was kann man über die RNase E sagen?
- major endo-RNase in Gram-negative bacteria, essential
- one of the largest proteins in E. coli (118 kDa) - hydrolytic phosphodiesterase - cleavage of single stranded AU rich region
- ist ein Tetramer: Dimer aus Dimeren
RNase E can interact with the cell membrane and a variety of different proteins
RNases, Hfq, metabolic enzymes (moonlighting proteins) …
RNase E functions as an assembly platform for the formation of the E. coli RNA degradosome
Was kann man über die RNase III sagen?
Ist eine double Strand specific RNase
- widespread in eubacteria
- orthologous in yeast, fly
plant, worm and human - 226 aa (26 kDa), homodimer - ds specific, Mg2+ dependent - not essential in E. coli but
mutants grow slower
Welche Funktionen hat die RNase III
- maturation of ribosomal RNAs
- degradation of sense/antisense
RNA duplices - host defense (phages and
horizontal gene transfer) - initiation of RNA degradation
of specific mRNA targets
Wie wird RNase III reguliert?
- constitutive expressed
- cleavage of own mRNA initiates
RNA degradation by RNase E
» negative autoregulation - activation by T7 protein kinase
» efficient T7 mRNA maturation
Welcher Unterschied ist zwischen Exo- und Endoribonuclease?
Exo: Spalten am 3’ oder 5’ Ende der RNA
Endo: Spalten inmitten der RNA
Exonukleasen
: Spaltung von den Enden der Nukleinsäurekette aus
Endonukleasen
: Spaltung innerhalb der Nukleinsäurekette Desoxyribonukleasen
: Spaltung von DNA
Ribonukleasen
: Spaltung von RNA
Welche katalytischen Mechanismen hat RNase III
Initial contact between dsRNA and either of the two RNA binding domains
Engagement with the dimeric catalytical domain
Binding of the second RNA binding domain
»_space; Precatalytic complex
Cleavage at both catalytic domains (Mg2+ and H2O are necessary)
Disengagement of the products from the RNase III domain
Release of the products from both RNA binding domains
Was gibt es über Exoribonucleasen zu sagen?
Ribonucleases that remove terminal nucleotides from either 5’ or 3’ end
5’-3’ exoribonuclease
3’-5’ exoribonuclease
Phospho- or hydrolytic enzymes
Processive
non-specific binding activity
1D-scanning/sliding
degrading enzymes
Distributive
no binding of non-targets
random 3D-diffusion
maturating enzymes
Wie viele RNase-Klassen gibt es?
6
Wie viele Exoribonucleasen gibt es in E.coli?
8
All 3’-5’ exoRNases
3 main exoRNases (RNaseII, RNase R, RNPase)
All degrade RNA in 2-5 nt fragments
All require ssRNA regions > 7-8 nt
RNase II and PNPase stop degradation 8 nt downstream from stem-loops
RNase R is only RNase that can degrade structured RNAs
Wie kommen die Triphosphase weg, die zum Schutz dienen?
durch RNA Pyrophosphohydrolase (RppH)
2017:
Sequential removal of γ- and β-phosphate (Luciano et al., Mol Cell)
First removal of γ-phosphate by an unknown enzyme
» 5‘-di-phosphate
5‘-di-phosphate is optimal RppH substrate
Wie wird man den den StemLoop am Ende der mRNA los?
Poly-A-Polymerase
Schützt die Polyadenylierung die mRNA in Prokaryoten?
Ne, sie favorisiert die Degradierung der RNA
> > Poly-A-tail : bodyguard in eukaryotes, scavenger in bacteria
Was ist die tmRNA?
1978 discovered (10S RNA) 1995 function detected
Encoded on the ssrA gene ~ 500 molecules / cell
1-10 % of ribosomes
Act as tRNA and as mRNA Interacts with SmpB protein
(encoded on the ssrB gene)
facilitates aminoacylation
mediates ribosome binding
protects against degradation
ssrA/ssrB pairs in all bacteria
essential only in some species
(Neisseria and Mycobacterium)
Required for full virulence in Salmonella
Wo findet die “Haupt” RNA Degradierung statt?
Im Degradosome, welches ein Multiproteinkomplex ist, der die wichtigsten endo- und Exoribonukleasen hat. Ist an der Membran attached
