Transcript Transcription of viral DNAs. Lecture 13 Flint et al. pp

Transcription of viral DNAs.
Lecture 14
Flint et al. pp. 253 – 277.
General points
• For RNA viruses, most viral mRNAs are
synthesized by Viral RDRP
• For DNA viruses, viral mRNAs are generally
synthesized by cellular RNA polymerases.
– Some exceptions: e.g. Poxviruses have their own
DNA-dependent RNA polymerase.
• Transcription and expression of viral genes
occurs in a strictly defined, reproducible
sequence.
• Generally:
– Genes for viral enzymes and regulatory proteins
are transcribed early in infection.
– Genes for structural proteins are transcribed
later.
• The relative simplicity of viral systems
has enabled their use in elucidating the
general properties of cellular RNA
transcription
• What we know about transcriptional
control is based on virology.
• Therefore, we must first digress back
to cellular transcription.
Cellular transcription
• DNA dependent RNA polymerase
• Cells contain 3 DNA dependent RNA polymerases (see
Table 8.1)
• RNA pol I: transcribes pre-rRNA; no known viral
templates
• RNA pol II: transcribes pre-mRNA & snRNA:
polymerase for most viral DNAs.
• RNA pol III: transcribes pre-tRNAs, 5S rRNA, U6
snRNA; polymerase for some viral DNAs.
Finding the right place to start
• The transcriptional machinery must:
– Be directed to initiate transcription at the correct
location on a DNA template (the transcriptional start
site).
– elongate through the entire gene
– Be directed to terminate transcription at the correct
location.
• All of these functions require the assistance of
– Cis-acting sequences along the DNA
– Trans-acting factors (accessory proteins)
Nuclear localization
• DNA dependent RNA polymerases located in
nucleus
• Viruses that usurp these functions must localize
their templates for this machinery into the
nucleus.
• Three variations on this theme (Table 8.2)
1. Viral genome looks like a chromosome:
-Associated with host-cell nucleosomes or viral
proteins that look like nucleosomes.
e.g. Papilloma- and Polyomaviruses
2. Viral ssDNA converted to dsDNA in viral particle.
dsDNA then imported into nucleus.
e.g. hepadnaviruses
3. Viral RNA reverse transcribed in viral particle. cDNA
imported into nucleus and integrated into genome.
e.g. Retroviruses.
Transcription by RNA Pol. II.
• At least 40 proteins required: Pol. II itself + accessory
proteins.
• Accurate transcription initiated at the promoter.
• Promoter + additional DNA sequence that controls
transcription = Transcriptional control region (TCR).
– The adenovirus type 2 major late promoter was the first TCR ever
recapitulated in vitro.
• Initiation is a multistep process:
–
–
–
–
Promoter recognition by RNA Pol. II
Formation of open initiation complex (unwinding)
Promoter clearance
3’ movement of complex away from promoter
Transcription
by RNA Pol.
II.
Fig 8.2
Promoters (Fig. 8.1)
Core promoters
• Contain all the information necessary for
recognition of initiation start site.
• Direct RNA Pol. II complex to begin
transcription.
• Contain TA-rich “TATA box” sequences.
– Thermodynamically easy to unwind.
– Located 20 – 35 bp upstream (5’) of start site.
• Contain Initiators
• Sort sequences
• Specify accurate but inefficient transcription
Promoters (Fig. 8.1)
Regulation of Pol. II
transcription
Transcription must be regulated: genes must be
turned on and off in temporal patterns
• Viral gene expression: early and late genes
• Transcriptional regulation is controlled by:
– Cis-acting sequences in DNA – both local and distal
– Trans-acting factors – both protein and RNA
• Trans-acting factors specifically bind to cisacting sequences to either
– activators stimulate transcription
– repressors prevent transcription
An enormous number of sequencespecific transcriptional regulators.
Basic properties (Fig. 8.7)
Modular organization – built from discrete structural
and functional domains.
• DNA binding module – targets protein to a specific
DNA sequence
• Activation (or repression) domain – interacts directly
or indirectly with the RNA polymerase.
• Dimerization domain – activation tends to require
interacts with other transacting factors. Provides a
way to fine tune activity.
– Homo-dimer: interaction of two copies of the same factor
– Hetero-dimer: interaction of two different factor.
Modular organization of
sequence-specific
transcriptional regulators
Example: Regulation of HIV-1 transcription by
host and virus encoded factors.
The HIV-1 transcriptional control region:
• Located in the 5’ region of the proviral genome.
• Divided into 3 enhancer regions.
– Promoter: Contains binding sites for TFIId and SP I factors:
recruit RNA Pol II to promoter.
– Core enhancer: binding sites for Nf-kB and Ets-1. These proteins
are only active in growing T-cells, i.e. those exposed to antigen.
– Upstream enhancer region: Binding sites for Ets-1, Gata-3, Lef, NfIL6. These proteins are only active in hematopoietic cells: helps to
narrow the specificity of transcription.
Transcriptional control by viral transacting factors: Tat and Tar (Fig. 8.13).
• By itself, RNA Pol II does not elongate well away
from the transcriptional control region: this
provides a way to downregulate (negatively control)
HIV replication.
• This is called poor processivity.
• To overcome this, the virus produces two transacting factors:
– Tar: an RNA trans-acting factor
– Tat: a protein trans-acting factor
• These two factors work synergistically to recruit
other host proteins that stimulate and enhance the
processivity of the elongating RNA Pol II complex.
Transcriptional control by viral trans-acting
factors: Tat and Tar (Fig. 8.13).
DNA virus transcriptional
programs
• Viral proteins act to either enhance or
repress transcription of viral genes in
an orderly manner.
DNA virus transcriptional
programs
• SV40: Genome contains 2 transcriptional units:
Early and Late.
• Early unit encodes the Large T antigen.
• Large T antigen acts to enhance transcription of
the late unit, leading to virus production.
DNA virus transcriptional programs
•
•
•
•
•
•
Adenovirus type 2: Has 3 transcriptional units:
Immediate early, early, and late.
E1A protein produced by immediate early unit
E1A activates early unit, represses late unit.
E2 protein encoded in early unit.
E2 activates transcription of the late unit.