Cytoplasm. Nuclear pores. AAAAAA. AAAAAA. DNA. Transcription. RNA. RNA Processing. G. G. mRNA. Export. Nucleus. Eukaryotic mRNA Transcripts are Processed. Transcription Start Site. 3’ UTR. 5’ UTR. Introns. 5’. 3’. Int. 1. Int. 2. Exon 1. Exon 2. Exon 3. Promoter/
Structure of eukaryotic and prokaryotic mRNAs:
• rRNAs are believed to play a catalytic role in protein synthesis.
• After removal of 95% of the ribosomal proteins, the 60S subunit can catalyze formation of peptide bonds.
• Ribosomal proteins are now believed to help fold the rRNAs properly and to position the tRNAs.
the mRNA is present on small subunit.
(P & A) bind tRNAs on large subunit.
Many RNA Viruses have capped genomic RNAs similar to eukaryotic host mRNAs
• Most eukaryotic mRNAs are capped at the 5’ end during nuclear processing.
• The terminal 5’ phosphate is first removed by a 5’ triphosphatase.
• Guanyltransferase transfers GMP from GTP to the 5’ end of the mRNA to add the GpppN cap structure.
• The 5’ terminal inverted G residue is then modified by methylation.
• Many RNA viruses replicate in the cytoplasm and must use a viral dependent capping mechanism supplied by the RNA-Dependent-RNA Polymerase.
• The Cap structure, m7GpppN, is most common in viral and mammalian mRNAs.
An initiation complex forms at the cap with the 40S ribosomal subunit and other translation initiation factors.
The 40S complex then scans down the 5’ untranslated region to the first AUG codon.
A GTP hydrolysis step by eIF5 triggers GDP binding of eIF2 and release of initiation proteins.
The 60S subunit joins the complex and the 80S ribosome initiates translate the ORF.
Ribosome selects aminoacylated tRNA
eEF1a and GTP are bound to aminoacylated tRNA
Ribosome catalyzes formation of a peptide bond
Translocation is dependent on eEF2 and GTP hydrolysis
Many ribosomes may translate mRNAs simultaneously on the same strand.
Type I-entero and rhinoviruses (poliovirus)
Initiation codon is located past the 3’ end of the IRES
40S binds to IRES scans to AUG
Type II-cardio and apthoviruses (EMCV)
Initiation codon is at the 3’ end of the IRES
40S binds at or near AUG no scanning occurs
Type III- hepatitis A virus
initiation codon is located past the 3’ end of the IRES
requires all of initiation proteins, including eIF4E
Type IV- hepatitis C virus
The 3’ end of the hepatitis C virus IRES extends
beyond the AUG codon
Type V-cricket paralysis virus
IRES ends at the initiation codon, although it is not an AUG codon, no initiation factors are required
initiation codon is placed at the A site instead of the P
Encephelomyocarditis virus (EMCV)
Cricket paralysis virus
Picornaviruses- Entire (+) sense RNA genome is translated into a single large polyprotein. Processing is carried out by two virus encoded proteases 2A pro and 3C pro.
Flaviviruses- Viral precursor proteins are processed by cellular proteases. The (+) sense RNA genome is translated into a polyprotein precursor processed by viral serine protease and by host signal peptidase.
Potyvirus group of plant viruses- Potato virus Y and tobacco etch virus contain a (+) sense genome RNA of around 10,000 bases which has a single open reading frame. This polyprotein is processed by viral encoded proteases.
Although majority of eukaryotic mRNAs are monocistronic, some viral mRNAs encode overlapping reading frames. The first start site is in a poor context, some ribosomes can bypass it and initiate at the second AUG, which has a better context. This will result in translation of two different proteins.
Rare in eukaryotes, but very common in prokaryotic cellular and viral mRNAs. Some eukaryotic mRNAs contain upstream AUG codons that terminate before the downstream reading frame. The upstream open reading frames may be translated, with reinitiation occurring at the downstream open reading frame.
In influenza B virus mRNA, M2 initiation codon is part of the termination codon for M1 protein. M2 synthesis is not efficient and dependent on M1 synthesis
Suppression of termination occurs during translation of may viral mRNAs as a means of generating a second protein with extended carboxy terminus. In retroviruses, gag and pol genes are encoded by a single mRNA and separated by an amber termination codon UAG. Translational suppression of the amber codon allows synthesis of the gag pol precursor.
A similar strategy is used by tobacco mosaic virus to translate its replicase proteins.
Translation suppression is mediated by suppressor tRNAs that can recognize termination codons and insert a specific amino acid. The nucleotide sequence 3’ of the termination codon also plays an important role in the efficiency of translational suppression.
Ribosomal frameshifting is a process in which ribosomes move to a different reading frame and continue translation in that reading frame.
It was discovered in cells infected with Rous sarcoma virus and has since been described for many other viruses including other retroviruses, (+) strand RNA viruses and herpes simplex virus.
Requires a “slippery” sequence X-XXY-YYZ (in Rous sarcoma virus A-AAU-UUA) and an RNA secondary structure called a pseudoknot five to eight nucleotides downstream.
Two tRNAs in the zero reading frame slip back by one nucleotide to the –1 phase and each tRNA base pairs with the mRNA in the first two nucleotides of each codon.
As a result of the frameshift a Gag-pol fusion is produced at about 5% of the level of Gag protein.
Rous sarcoma virus mRNA encodes Gag and Pol proteins that overlap in a –1 reading frame
Interferons are produced in response to viral infection as part of the rapid innate immune response
Interferons bind to cell surface receptors and activate transcription of antiviral genes
Two interferon induced genes encode RNase L and protein kinase RNA-activated (Pkr)
RNase L degrades RNA
Pkr phosphorylates eIF2a, inhibiting translation initiation
Pkr is a serine threonine kinase composed of an N-terminal regulatory domain and a C-terminal catalytic domain
Pkr is activated by the binding of dsRNA to two dsRNA binding motifs at the N-terminus of the protein.
Viruses use at least five different mechanisms to block Pkr activation or to stop activated Pkr from inhibiting translation
• inhibition of dsRNA binding-
adenovirus VA RNA binds Pkr blocks its activation by dsRNA
• vaccinia virus E3L protein sequesters ds RNA
• inhibition of Pkr dimerization
influenza virus P58
hepatitis C virus NS5A
• inhibitors of kinase function-
vaccinia virus K3L protein has homology to N- terminus of eIF2-a