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Eukaryotic mRNA Transcripts are Processed
Cytoplasm
DNA
Transcription
RNA
RNA
Processing
mRNA G
G
AAAAAA
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Nucleus
AAAAAA
A “Simple” Eukaryotic Gene
Transcription
5’ UTR
Start Site
3’ UTR
Introns
5’
Exon 1Int. 1
Promoter/
Control Region
3’
Exon 2 Int. 2 Exon 3
Exons
Terminator
Sequence
Transcription
5’
Exon 1Int. 1
Exon 2 Int. 2 Exon 3
Unprocessed RNA Transcript
3’
Processing of eukaryotic mRNA
5’ UTR
G
5’ Cap
3’ UTR
Protein Coding Region
Exon 1 Exon 2 Exon 3
AAAAA
3’ Poly A Tail
• RNA processing achieves three things:
1) Removal of Introns
2) Addition of a 5’ cap
3) Addition of a 3’ tail
 The mRNA then moves out of the nucleus and
is translated in the cytoplasm.
Translational control
Structure of eukaryotic and prokaryotic mRNAs:
Model of eukaryotic ribosome
• 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.
•Small & large ribosomal
subunits.
•A Binding site for
the mRNA is present on
small subunit.
•Two binding sites
(P & A) bind tRNAs on
large subunit.
–P site – holds the tRNA
carrying the growing
polypeptide chain.
–A site – holds the tRNA
with the next AA to be
added.
Ribosome Structure
• Ribosomes hold the
mRNA and tRNAs together
and connect the amino
acids at the A site to the
growing polypeptide.
Structure of tRNA
• Aligns each amino acid
with the corresponding
codon
• 70-80 nt long
• 3’ end has the 5’- CCA
sequence to which aa are
linked
• The opposite end
contains the anticodon
loop
• Contains modified
bases
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.
Three distinct stages of translation
Initiation
•Rate limiting step
•Requires hydrolysis of ATP and GTP
•Results in formation of a complex containing the
mRNA, the ribosome and the initiator Met-tRNA
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
5’ end (cap) dependent initiation:
• The first step is the recognition of the 5’ cap by
eIF4F, which consists of three proteins, eIF4E, eIF4G
and eIF4A.
• Cap binding protein, eIF4E, binds to cap
• The N-terminus of eIF4G binds eIF4E and the Cterminus binds eIF4A
• The 40S subunit binds to eIF4G via eIF3
Cap-Dependent Initiation of Protein Synthesis in Eukaryotes
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.
Elongation
eEF1a
GTP
Ribosome selects
aminoacylated tRNA
eEF1a and GTP are bound to
aminoacylated tRNA
P A
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.
eEF2
GTP
Termination
•Translation is terminated
at one of three stop codons
(UAA, UAG & UGA).
• Termination codon at the
A site is recognized by the
release factor instead of a
tRNA
• The release factor binds
the termination codon
• The peptide chain is then
released followed by
dissociation of the tRNA
and the ribosome
Closed loop model:
• 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
5’end (cap) independent initiation:
Poliovirus
• mRNAs of picornaviruses lack 5’ cap
• Ribosomes bind internally rather than at the mRNA 5’ end
• 5’ end of poliovirus RNA promotes internal binding of 40S subunit at
internal ribosome entry site (IRES)
• In poliovirus infected cells eIF4G is cleaved, inactivating translation
of cellular mRNAs
•The initiation in the IRES does not depend on the presence of a cap
structure, but requires C-terminal fragment of eIF4G to recruit the 40S
subunit through interaction with eIF3.
Five different types of IRES sequences are found on viral RNAs:
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
site
Four types
of IRES:
polivirus
Encephelomyo
carditis virus
(EMCV)
Hepatitis C
Cricket
paralysis
virus
Hepatitis C virus IRES:
• 40S ribosomal subunit binds directly to
Hepatitis C virus IRES in the absence of most
initiation factors
• A dramatic change in the conformation of the
40S subunit occurs when it binds Hepatitis C
virus IRES setting the AUG at the P site
• eIF3 also binds to the Hepatitis C virus IRES
Bicistronic mRNA assay for IRES elements
Viral translation strategies
Polyprotein synthesis
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.
Polyprotein processing in enteroviruses and flaviviruses
Poliovirus:
Flavivirus:
Leaky scanning
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.
Reinitiation
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.
Reinitiation of translation in influenza B virus
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
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.
Suppression of termination codons in
alphaviruses and retroviruses
Pseudonot
alphavirus
retrovirus
Ribosomal frameshifting
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 AAAU-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.
Ribosomal frameshifting
Rous sarcoma virus
mRNA encodes Gag
and Pol proteins that
overlap in a –1 reading
frame
REGULATION OF TRANSLATION DURING VIRAL INFECTION
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.
Model of activation of Pkr
Viral regulation of Pkr
Viruses use at least five different mechanisms to block Pkr
activation or to stop activated Pkr from inhibiting
translation
• inhibition of dsRNA bindingadenovirus 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 functionvaccinia virus K3L protein has homology to Nterminus of eIF2-a
Regulation of eIF4F activity by different viruses