RNA Degradation in Bacteria

Question 1

Was ist die a. doubling time b. average mRNA half-lives von 1.E.coli 2.S.cerivisiae 3.Human

Answer 1

1.a. 20-60min b.3-5min
2.a. 180min b.22min
3.a. 16-24h b.10h

Question 2

Was sind die Schritte zur RNA Halbwertszeit Messung?

Answer 2

1. Kultivierung
2. Rifampicin addition
3. sampling
4. RNA Preparation
5. Northern blotting/ qRT-PCR
6. Detection
7. Reprobing
8. Detection
9. Normalization
10. Calculation

Question 3

Was ist Riampicin?

Answer 3

polyketide Antibiotika, produziert von Soil Bakterien, die RNAP inhibieren

Question 4

Was sind Ribonucleasen? RNasen?

Answer 4

• Nukleasen, die RNA zersetzen
• Spaltung von Phosphordiesterbindungen in RNA Substraten

Question 5

Wie viele Enzymklassen und Subgruppen gibt es?

Answer 5

2 und 2
 EC2.7 phosphorolytic enzymes
 EC3.1 hydrolytic enzymes
und Subgruppen:
 endoribonuclease
 exoribonuclease

Question 6

Welche Funktionen haben RNasen?

Answer 6

 mRNA turnover
 maturation of stable RNAs  degradation of replication primer
 sRNA mediated gene regulation
 defense against horizontal gene transfer

Question 7

Was ist die RNA Reifung

Answer 7

 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

Question 8

Was ist die RNA Degradierung?

Answer 8

 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

Question 9

Wie läuft das rRNA Processing der rRNA Reifung ab?

Answer 9

 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

Question 10

Wie läuft die tRNA Reifung ab?

Answer 10

 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

Question 11

Was schützt eine mRNA vor den Abbau von Endoribonucleasen?

Answer 11

Triphosphate an einem Ende; Stem Loop Ende

Question 12

Was sind die Endonucleasen?

Answer 12

I. Internal cleavage by an endonuclease
 RNase E (E. coli)
 RNase Y (B. subtilis)
 RNase II

Question 13

Was kann man über die RNase E sagen?

Answer 13

• 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

Question 14

Was kann man über die RNase III sagen?

Answer 14

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

Question 15

Welche Funktionen hat die RNase III

Answer 15

• 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

Question 16

Wie wird RNase III reguliert?

Answer 16

• constitutive expressed
• cleavage of own mRNA initiates
  RNA degradation by RNase E
  » negative autoregulation
• activation by T7 protein kinase
  » efficient T7 mRNA maturation

Question 17

Welcher Unterschied ist zwischen Exo- und Endoribonuclease?

Answer 17

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

Question 18

Welche katalytischen Mechanismen hat RNase III

Answer 18

 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
&raquo_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

Question 19

Was gibt es über Exoribonucleasen zu sagen?

Answer 19

 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

Question 20

Wie viele RNase-Klassen gibt es?

Answer 20

6

Question 21

Wie viele Exoribonucleasen gibt es in E.coli?

Answer 21

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

Question 22

Wie kommen die Triphosphase weg, die zum Schutz dienen?

Answer 22

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

Question 23

Wie wird man den den StemLoop am Ende der mRNA los?

Answer 23

Poly-A-Polymerase

Question 24

Schützt die Polyadenylierung die mRNA in Prokaryoten?

Answer 24

Ne, sie favorisiert die Degradierung der RNA

> > Poly-A-tail : bodyguard in eukaryotes, scavenger in bacteria

Question 25

Was ist die tmRNA?

Answer 25

 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

Question 25

Wo findet die “Haupt” RNA Degradierung statt?

Answer 25

Im Degradosome, welches ein Multiproteinkomplex ist, der die wichtigsten endo- und Exoribonukleasen hat. Ist an der Membran attached