14.Transcription Stratergies: DNA templates

Transcription of viral DNAs.
See Text (Flint et al), pp. 253 – 277.
Lecture 14
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.
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
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.
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:
1. Be directed to initiate transcription at the correct location on a DNA template (the
transcriptional start site).
2. elongate through the entire gene
3. 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
The DNA dependent RNA polymerases are located in the nucleus
Viruses that usurp these functions must localize their templates for this machinery into
the nucleus.
Three variations on this theme (Table 8.2)
 Viral genome looks like a chromosome: e.g. associated with host-cell
nucleosomes or viral proteins that look like nucleosomes. e.g. Polyomaviruses
 Viral ssDNA converted to dsDNA in viral particle. dsDNA then imported into
nucleus. E.g. hepadnaviruses
Viral RNA reverse transcribed in viral particle. cDNA imported into nucleus and
integrated into genome. e.g. Retroviruses.
Transcription by RNA Pol. II. (Fig. 8.2)
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.
The adenovirus type 2 major late promoter was the first TCR ever recapitulated in vitro.
Initiation is a multistep process:
1. Promoter recognition by RNA Pol. II
2. Formation of open initiation complex (unwinding)
3. Promoter clearance
4. 3’ movement of complex away from promoter
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.
o Thermodynamically easy to unwind.
o Located 20 – 35 bp upstream (5’) of start site.
 Sort sequences
 Specify accurate but inefficient transcription
Regulation of Pol. II transcription
Transcription must be regulated: genes must be turned on and off in temporal
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 cis-acting sequences to either
 Stimulate transcription – activators
 Prevent transcription – repressors
An enormous number of sequence-specific 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
 Dimerization domain – activation tends to require interacts with other transacting
factors. Provides a way to fine tune activity.
o Homo-dimer: interaction of two copies of the same factor
o Hetero-dimer: interaction of two different factor.
Example: Regulation of HIV-1 transcription by host and virus encoded factors.
Transcriptional control by host-encoded factors (Fig. 8.11; Table 8.4)
HIV-1 transcriptional control region:
 Located in the 5’ region of the proviral genome, beginning approximately 200 bp
5’ of the Gag gene translational start site.
 Divided into 3 enhancer regions.
o Promoter: Contains binding sites for TFIId and SP I factors. Binding of
these factors to DNA recruits RNA Pol II to promoter.
o Core enhancer: binding sites for Nf-B and Ets-1. These proteins are
only active in growing T-cells, i.e. those exposed to antigen.
o Upstream enhancer region: Binding sites for Ets-1, Gata-3, Lef, Nf-IL6.
These proteins are only active in hematopoietic cells: helps to narrow the
specificity of transcription.
Transcriptional control by viral trans-acting factors: Tat and Tar (Fig. 8.13).
 By itself, RNA Pol II does not elongate away well from the transcriptional control
region: this provides a way to downregulate (negatively control) HIV
 This is called poor processivity.
 To overcome this, the virus produces two trans-acting 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.
DNA virus transcriptional programs (SV40 and Adenovirus type 2. Fig. 8.14)
Viral proteins act to either enhance or repress transcription of viral genes in an orderly
 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
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.