RNA Synthesis and Regulation from DNA Templates: DNA virus and Retroviruses Flashcards
Where does transcription, translation, and genome replication of RNA viruses occur?
Transcription: Cytoplasm
Translation: Cytoplasm
Replication: Cytoplasm
Where does transcription, translation, and genome replication of DNA viruses occur?
Transcription: Nucleus
Translation: Cytoplasm
Replication: Nucleus
Where does transcription, translation, and genome replication of retroviruses and para-retroviruses occur?
Transcription: Nucleus
Translation: Cytoplasm
Replication: Nucleus
What is special about Orthomyoxiviridae’s transcription and genome replication?
Transcription: Nucleus
Replication: Nucleus
What is special about Poxviridae’s transcription, translation and genome replication?
Everything is in the cytoplasm
What are the four key features of transcription in eukaryotes?
1) Confined to the nucleus
2) Three distinct types of RNA polymerases and associated factors
3) RNA processing
4) Combinatorial regulation
What are three types of RNA polymerases and their dependency?
1) RNA POL I –> DNA-dependent RNA polymerase I
2) RNA POL II –> DNA- dependent RNA polymerase II
3) RNA POL III –> DNA-dependent RNA polymerase III
What is special about RNA POL II?
Most viruses use POL II
What is the composition or core polymerases of RNA POL I? (5)
RPA1, RPA2, RPC5/RPC9, RPB6, and 9 others
What is the composition or core polymerases of RNA POL II? (5)
RBP1, RBP2, RPB3, RPB6, and 7 others
What is the composition or core polymerases of RNA POL III? (5)
RPC1, RPC2, RPC5, RPB6, and 11 others
What cellular RNAs are synthesized from RNA POL II?
Pre-mRNAs, pre-miRNAs, four snRNAs (U1, U2, U4,U5)
What cellular RNAs are synthesized from RNA POL III?
Pre-tRNAs, 5srRNA, snRNA (U6)
What viral RNAs are synthesized from RNA POL I?
None identified
What viral RNAs are synthesized from RNA POL II?
Pre-mRNAs, mRNAs, pre-miRNAs, and genomic RNA
What viral RNAs are synthesized from RNA POL III?
Ad2 VA-RNAs, EBV EBER RNAs
What does it mean for mRNA transcription in eukaryotes to be combinatorial?
1) Ensures a gene is transcribed only when it needs to be (avoid accidental transcription from off-target gene)
2) Sharing of a finite set of transcription activators among many genes
3) Offers fine tuning and regulation of gene expression via the combinatorial effects of different combinations of transcription factors
What are the common DNA binding motifs?
Zinc finger
Helix-turn- helix
Basic helix-loop-helix
Homeodomain
What is the DNA dimerization domain?
Leucine zipper helix
What are the features of a DNA activation domain?
Acidic
Rich in glutamate
Proline and isoleucine
What is the limiting step in transcription?
Initiation
What was the dilemma with RNA splicing?
Precursors of mRNAs are heterogenous in size (hnRNAs) and these hnRNAs are much longer than mature mRNAs
What do hnRNAs have?
They have preserved sequences at both the 5’ and the 3’ ends after treatment with RNAse1
What happened when viral genomic DNA was hybridized with hexon mRNA?
Produces three loops in the mRNA (introns)
What is mRNA splicing?
Introns are spliced out of pre-mRNA to get mature mRNA
Why is alternative splicing important (4)?
1) Allows multiple mynas and proteins to be produced from a single gene
2) Different isoforms of proteins have different functions, which are important for gene regulation
3) Enables the mix and match of sequence modules and functional domains
4) Maximize the coding capacity of limited genomes
What does HSV1 produce?
The protein VP16
What are the functions and properties of HSV1 ? (2)
1) Stimulates transcription from IE promoters via its potent acidic activation domain
2) Achieves promoter specificity by interacting with two cellular factors, Oct-1 and Hcf
What does SV40 produce?
The LT antigen
What are the functions and properties of SV40? (4)
1) Stimulates late gene transcription
2) Binds to Top, TfIIB, and Tef-1 via different domains
3) no activation domain
4) May stabilize initiation complex
What is the transcription activator for HSV-1?
ICP4
What is the function of ICP4 in HSV-1?
1) Induces transcription from E and L promoters
2) Represses IE transcription (auto-repression)
What does HSV-1 contain? (5)
IE, EE, LE, LL, TL genes
What is expressed as late genes in HSV-1?
Essential proteins required for entry and DNA replication are expressed as late genes and packaged into the virion.
These are the tegument proteins: VP16, VHS, ICP27
What happens when HSV-1 enters the cell?
When HSV-1 enters the nucleus of the host cell, the linear genome is circulated by cellular enzymes
What is special about the viral transcripts in HSV-1?
They lack introns, so individual genes need to be transcribed individually
ICP27 can actually block the splicing of host pre-mRNA because they lack introns, so no splicing occurs
What is the cycle of infection for HSV-1?
Switches between latency and lytic infection
In latency of viruses what happens?
In latency, viral mRNAs are not produced, so no viral DNA replication and no virion formation
There are three latency associated transcripts detected in infected neuronal cells
Research demonstrates the production of 16 miRNAs, most of which are derived from the LAT region
What are the RNA POL III transcriptions for HAV-2? What is the function?
VA-RNA1, VA-RNAII
Prevent activation of dsRNA-dependent protein kinase
What are the RNA POL III transcriptions for EBV? What is the function?
EBER-1 and EBER-2
Always made in latently infected cells and maintain latency
What are the RNA POL III transcriptions for Herpes salmiri-2? What is the function?
HSVR 1- 5
Degrade certain cellular mRNAs
What are the RNA POL III transcriptions for Moloney murine leukaemia virus? What is the function?
Let Stimulates transcription of specific cellular genes (CD4 and class I MHC genes
What is the TATA Box?
Binding by TBP, followed by the assembly of the pre-initation complex, basal transcription
What is +1?
This is the transcription start site the first nucleotide being synthesized. It will always be a purine
What is the coding DNA strand?
5’ –> 3’ and is the strand that has the same sequence as its transcript RNA
What is the template DNA strand?
3’ –> 5 and is the strand used as the template to make RNA copies
What are transcription regulators?
Activators, co-activators, and repressors
They bind regulatory sequences and modulate transcription through a mediator complex (looping model of transcription)
What are the distant regulatory sequences?
Enhancers and silencers
Far from transcription start site
What makes up the core promoter?
TATA sequence and the initiator sequence
What is the initiator sequence?
Where transcription starts
What makes up the promoter?
core promoter and local regulatory sequences
What makes up the transcriptional control region?
Distant regulatory sequences and the promoter
What happens in the RNA polymerase during transcription and after?
During transcription there is a bend in DNA, and DNA re-anneals after transcription
How does DNA enter and exit the polymerase?
DNA enters the polymerase downstream and exits upstream
What makes up the RNA polymerase?
A wall, a pore, a funnel, ribonucleoside enter site and a groove
What are the commonly used inhibitors of transcription?
Antinomycin D
alpha- Amanitin
What is guanylyl-transferase associated with and why?
Guanylyl transferase is associated with CTD of Pol II to ensure each mRNA is capped as it is transcribed
How is 5’ capping carried out?
On a short (20-30 nt) nascent pre-mRNA transcribed by POL II
What is the 5’ cap?
It is a special molecule of 7 methyl-guanosine linked to the 5’ terminal nucleotide of pre-mRNA via a 5’- 5’ triphosphate linkage
What is the 5’ cap’s function?
To signal and protect mRNAs for translation
How was the 5’ cap discovered?
In 1970 in a reovirus and vaccinia virus
What is the 5’ cap bound by?
The 5’ cap is bound by the cap-binding complex.
Once the cap is added, the guanylyl-transferase dissociates and CBC joins
What and where are the promoters for adenovirus transcription and RNA splicing?
5 on the top strand: E1A, E1B, E3, IX, and major late
3 on the bottom strand: E4, E2A/E2B, and IVa2
How does adenovirus transcription work?
11 polyadenylation sites
13 families of viral genes, encoding 50 proteins
The major late promoter is present and very active during late infection
How does adenovirus transcription termination occur?
There are 5 late promoters, and transcription can terminate anywhere from L1 to L5
What proteins do each of the late promoters encode?
L1: IIIa, 52/55k L2: V, PVII, penton (III) L3: 23K (protease), hexon (II), PVI L4: pVIII, 33K, 100K L5: fiber (IV)
How are sequences of introns recognized for splicing of pre-mRNAs?
1) 5’ splice site in exon 1: A/G A G
2) 3’ splice site in exon 2: G
3) Branchpoint in in the intron which is an A
What is constitutive splicing?
Let’s say you have exon 1 intron 1 exon 2 intron 2 exon 3.
Constitutive splicing you get: exon1-exon-2-exon 3
What is alternative splicing?
Let’s say you have exon 1 intron 1 exon 2 intron 2 exon 3
Exon skipping: exon1-exon 2
Alternative 5’ splice sites
Alternative 3’ splice sites
What is special about viral transcription control regions?
They resemble those of eukaryotic genes
What do the SV40 early, Ad2 early, and Ad2 late genes have in common?
They all contain the TATA box to ensure assembly of the POL II holoenzyme
Each promoter contains upstream DNA binding sites for multiple transcription activators encoded by the host cell
What do the early promoters for SV40 and Ad2 have?
They contain repeats of the same DNA sequences, even in reverse direction, to allow binding of multiple copies of the transcription activator
Where can transcription initiation begin?
Multiple positions in the Inr sequence
Explain the SV40 early gene
Has many Sp1 sequences (stimulatory protein 1) and a TATA box, and transcription initiation can start from many sites within the promoter region
Explain the Ad2 early gene
Has an Atf (activating transcription factor), two E2F sequences (E2 factor, which binds to the E2 gene promoter in adenoviruses) and a TATA box, and transcription initiation can start from many sites within the promoter region
Explain Ad2 late genes
Has a CpI site, and a Usf sequence (upstream stimulatory factor), and a TAT box. Transcription can only begin from one position in the promoter region
How does 289R and 243R stimulate RNA transcription?
1) Both isoforms recruit HAT (p300/Cpb), loosening up nucleosomes for transcription
2) Both isoforms bind pRb, releasing transcription activator E2F, activating expression of a wide range of genes, forcing cell entry to S phase
3) E2F activates E2 expression, producing proteins required for genome replication
4) 289R binds Med23 (part of mediator complex), Atf-2 and Sp1, inducing E3 and E4 transcription.
Why is VP16 important and where does it come from?
VP16 activates transcription in HSV and is an abundant tegument protein in the virion
How does VP16 activate transcription
VP16 activates transcription of IE genes through interaction with two cellular transcription factors: Oct-1 and HCF
Oct1 binds at the octane sequence (TAAT-GARAT) in promoters of IE genes
HCF binds VP16 in the cytoplasm and transports VP16 to the nucleus
VP16 bound with HCF associates with Oct-1, stimulating transcription via two mechanisms: assembly of initiation complex, and chromatin remodelling
How do retroviruses transcribe proviral DNA
Transcription control elements are located in the U3 region of LTR
No viral transcription factors, total reliance on the host cell factors
Transcription control regions are recognized by various common transcription factors in both avian and mammalian species
Avian and mammalian factors are homologs of each other
What is each region in proviral DNA and what do they produce in avian cells?
CArG region (2): EfII Y region (2): Yb-1
What is each region in proviral DNA and what do they produce in mammalian cells?
CArG region (2): Srf Y region (2): Cbf, Nf-y
What is each region in proviral DNA and what do they produce in third option?
CArG region (2): MADS box Y region (2): Cbf, Y box
Where are the retroviruses promoters and enhancers?
Promoter downstream from -100 bp
Enhancer upstream of -100 bp
If confused there is a figure
What is the domain structure of TAT? What does each domain structure do?
N-terminal - Cytosine rich - Core - Basic - C terminal
N terminal and Cytosine rich and Core: Stimulation of activation domain
Basic: RNA binding in vitro (NLS)
How does HIV-1 undergo transcription?
1) TAT enhances transcription of full length viral RNA through binding at TAR
2) TAR forms a SL structure in short nascent RNA transcript, impeding elongation of transcription
3) TAT recruits p-Tefb (cyclin T and Cdk9) and phosphorylates CTD of POL II, increasing full length viral RNA by 100-fold
How is Ad2 VA-1 RNA transcribed by RNA POL III?
A and B boxes are essential for efficient transcription of VA-1 gene
1) TfIIIC1 and TFIIIc2 bind to the intragenic promoter
2) TfIIIb binds TFIIIc- DNA complex and recruits RNA POL III
3) Transcription termination resembles that of tRNA genes
