Translational Strategies Flashcards
(25 cards)
What are the functions of the 5‘ cap of mRNAs?
The 5‘ cap marks the first exon and aids the splicing process
- essential for the transport from nucleus to cytoplasm (nuclear cap-binding proteins)
- protection gains 5‘-3‘ endonuclese activity
- protects viral RNA from antiviral defence mechanisms
- increases the efficiency of translation, targets preinitiation-complex
What enzymatic reactions are necessary for the capping of mRNA?
- Removal of terminal 5‘ phosphate by 5‘ phosphates
- Transfer of GMP to 5‘ end to add GpppN structure by guanyltransferase
- Methylation of 5‘ G residue
What are the 3 ways of viral mRNA capping?
- de Novo synthesis by cellular enzymes
- synthesis by viral enzymes
- cap snatching
Name the three unconventional capping pathways and their mechanism.
a. Rhabdoviridae-like:
b. Alphaviridae like
C. Orthomyxoviridae-like
Name the functions of the 3‘ prossesing of mRNA (PolyA).
- stabilisations of mRNA - protection against endonuclease
2. increase in translation efficiency
What are the special features of viral transcription?
- Many viruses use translation strategies to maximize the coding capacity of their genomes of limited size
- functional polycistronic mRNAs
- readthrough of stop codons
- polyproteins - Virus infection often results in modification of the host‘s translational machinery to translate selectively the viral mRNAs -„Host cell shutoff“
What are the four main sources of getting as much gene products out of one primary transcript?
-Poly protein processing
-initiation at different start codons
•Reinitiation
•leaky scanning
•internal ribosomal entry
•ribosomal shunting
-changing the translational reading frame
•splicing
•ribosomal frame shifting
•RNA editing
-translational readthrough of stop codons
What are the two forms of translation initiation?
A. 5’ end (Cap) dependent initiation
• The initiation complex binds to the 5’ cap structure and scans in a 5’ to 3’ direction until initiating AUG is encountered
B. Internal ribosome entry
• Initiation complex binds upstream of initiation codon (EMCV IRES, Poliovirus IRES or directly at the start codon (BVDV/HCV IRES; CrPV-IRES)
What are the steps of 5‘ cap dependent initiation?
- The first step is the recognition of the 5’ cap by eIF4F, which consists of three proteins, eIF4E, eIF4G and eIF4A.
- The cap binding protein eIF4E binds to the 5’ cap
- The N-terminus of eIF4G binds eIF4E and the C-terminus binds eIF4A
- The 40S subunit binds to eIF4G via eIF3
What is the advantage of the closed loop model of mRNA translation?
- The 5’ end dependent initiation is stimulated by the poly(A) binding protein Pabp1p, which interacts with eIF4G
- This interaction circularizes the mRNA and facilitates formation of the initiation complex
- Mechanism to ensure that only intact mRNA is translated
What can inhibit the initiation of translation?
- missing 5‘cap-structure no formation of the initiation complex and therefore no ribosome recruitment
- strong RNA secondary structure in 5 ́NTR
- long 5 ́NTR
- upstream AUG and open reading frames
- poor sequence context of the AUG codon (Kozak-rule)
What ist the molecular mechanism of PV 5‘cap independent translation? How does PV initiate translation?
- Complete shut off of cap-dependent translation of host cell mRNAs in cells infected with Poliovirus
- Poliovirus genomic RNA is efficiently translated under these conditions
eIF4G
molecular basis?
cleavage of eIF4G by Poliovirus encoded protease 2A
cap-independent translation of Poliovirus RNA: - IRES-mediated
- efficient translation after eIF4G cleavage
- Use of the C-terminal cleavage product
What are the four IRES types and their requirements for translation initiation factors?
Type-1(picornavirus): Initiation without eIF4E, scanning to get to startcodon
Type-2(picornaviruses): Initiation without eIF4E, startcodon directly after IRES - no scanning needed
Type-3(HCV-like): Initiation without eIF1, eIF1A, eIF4A, eIF4B and eIF4F - does not need initiation komplex, only 48S complex.
Type-4(dicistrovirus intergenic Region): initiation without eIFs or tRNAi, only needs 40S, part of the structure mimics tRNAi
How does the temporal regulation of NS2-3 cleavage in pestivirus(BVDV) work?
Binding or jiv promotes the autoprotease activity of NS2, NS2-3 is cleaved and RNA is replicated efficiently.
In the later phase of virus infection the cellular jiv pool is depleted as it is degradated in the NS2-jiv complex. Therefore NS2-3 is not cleaved and virion morphogenetic sets in.
Describe the mechanism of FMDV 2A/2B processing.
Addition of the ultimate 2A- residue
• Peptidyl-2A-tRNA is translocated to the P-site
• Ingress of prolyl-tRNA
• Prolyl-tRNA is unable to attack the peptidyl-2A-tRNA ester linkage
• Hydrolysis of the glycyl-tRNA ester bond and nascent peptide release
• Translocation of prolyl-tRNA to P site. Synthesis of the remaining peptide C- terminal of 2A would proceed as normal.
What are the four forms of the initiation at different start codons?
- Reinitiation: ribosomes stays engaged to mRNA after stop codon/ stop codon and start codon overlap
- Leaky scanning: ribosome reads through a start codon due to poor initiation context (kozak-rule)- initiation at downstream ORF
- Ribosome shunting: RNA structure leads to the transport of the initiation complex to a downstream ORF
- internal ribosomal entry site (IRES) - the ribosome binds to a RNA structure in the mRNA (cap independent) and starts translation there
What does the Reinitiation of translation depend on?
- sequence context of the uORF AUG
- RNA secondary structure between uORF and downstream ORF
- distance between the two ORFs
- viral factors
In what ways is the change of the Reading Frame in translation achieved?
- splicing
- RNA editing
- ribosomal frame shifting
How is the ribosomal frame shifting achieved? Explain for the case of retrovirus.
RNA signals: „slippery sequence“ followed by a pseudoknot or a stem-loop
A „slippery sequence“ forces the ribosome into the -1 reading frame for Coronavirus and most retroviruses (Yeast Ty1 +1) upstream of the stop codon
Retroviruses:
- Ribosome pausing because of downstream pseudoknot/stem-loop structure - Slippage of two tRNAs into -1 frame before/after peptidyl transfer
- each tRNA pairs with the mRNA in the first two nucleotides of each codon
- 3’ base of the 3’ codon is available for binding of next incoming tRNA
- free codon in mRNA resulting in „-1 reading-frame“ of the mRNA
What isotherm result of RNA editing in HDV?
- adenosine desamination of A leads to I
- during replication I basepairs with C (not A with U); mRNA contains Trp-codon not the stop codon - S-HDAg (Replication) replaced by L-HDAg (Assembly)
Name two examples for the read through of a stop codon (initiation termination).
- Mo-MLV (moloney murine leukemia virus): RNA pseudoknot slows ribosome down - translation of Gln (CAG) instead of stop codon (UAG)
- Sindbis Virus (Alphavirus): Opal Stop codon (UGA); nucleotide context UGAC- downstream C is important for„read through“ and suppression of translation termination (10% of cases)
Explain the inactivation strategies of the cellular eIF4F-complex by viral factors.
- A viral protease (polio: 2Apro, foot-mouth disease: L) cleaves the „bridge“ eIF4G, resulting in the los of the cap binding subunit of the translation initiation complex - no cap dependent translation.
- Dephosphorylation of 4E-bpI leads of the binding of the binding domain of eIF4E (Polivirus)
- dephosphorylation of eIF4E leads -no binding to eIF4G (Adenovirus, influenza virus)
What is the interferon (IFN)-effect?
• Virus-infected cells secrete IFN and IFNß (Type1 IFN)
• IFN generates antiviral state in non-infected cells
(ca. 300 IFN regulated genes), e.g. via RNAse L
• INF induces the synthesis of protein kinase R (PKR) - detects dsRNA/DNA
• viral dsRNA activates PKR; it then blocks translation initiation
• INF induces the synthesis of Mx protein; which inhibits viral replication e.g. of influenza viruses and other (-)-strand RNA viruses
What is the effect of eIF2alpha phosphorylation?
eIF2*GTP brings initiator tRNAi to the Initiation complex
When eIF2a is phosphorylated it binds irreversible to eIF2b and therefore can not be recycled. - inhibition of initiation
