Molecular Virology

1. What are the three types of RNA polymerases (RNApol)?

2. RNApol V- responsible for RNA interference

1. RNApol I- cellular pre-mRNA2. RNApol II- transcript proteins coding gene and mRNA3. RNApolIII- transcribe tRNA

1. RNApol IV- involved in post-transcriptional RNA processing

3. RNApol VI- essential for DNA replication

RNA polymerases are enzymes responsible for synthesizing RNA from a DNA template. In eukaryotes, RNApol I synthesizes ribosomal RNA (rRNA), RNApol II transcribes protein-coding genes and messenger RNA (mRNA), and RNApol III transcribes transfer RNA (tRNA) and other small RNAs. Therefore, options 1, 2, and 3 are incorrect as they do not accurately describe the functions of the different RNA polymerases.

Explanation

RNA polymerases are enzymes responsible for synthesizing RNA from a DNA template. In eukaryotes, RNApol I synthesizes ribosomal RNA (rRNA), RNApol II transcribes protein-coding genes and messenger RNA (mRNA), and RNApol III transcribes transfer RNA (tRNA) and other small RNAs. Therefore, options 1, 2, and 3 are incorrect as they do not accurately describe the functions of the different RNA polymerases.

2. What are the steps of RNA synthesis?

1. RNA polymerase does not bind to the promoter to initiate transcription.

2. Elongation of RNA occurs by adding DNA nucleotides.

1. PolII binds to promoter and begins initiation2. Elongates by adding NTP and creating a 5' nascent RNA3. Once processivity is initiated, full transcription gene occurs4. Termination occurs by RNApol falling off

3. The termination of RNA synthesis involves the addition of more nucleotides.

3. Initiation :1. ___ and ____ factors mediate this process2. _____ (__) is recognized by RNApolII on closed initiation complex3. ______ is opened and an abortive transcript is created by RNApol falling off4. ____ bind back on the initial transcribing complex and creates a nascent RNA..

1. Structural and regulatory factors2. Insulator region (A-rich)3. Transcription complex4. RNApol IV.

1. viral and cellular factors2. Core promoter (TATA and +1)3. Initiation complex4. RNApol I

1. Bacterial and fungal factors2. Enhancer region (GC-rich)3. Elongation complex4. RNApol II.

1. Environmental and genetic factors2. Exonuclease region (C-rich)3. Termination complex4. RNApol III.

The correct answer involves the interaction between viral and cellular factors during the initiation process, RNApolII recognition of the core promoter (TATA and +1), formation of the initiation complex leading to nascent RNA synthesis by RNApol I. The incorrect answers provide plausible but incorrect information related to various factors and processes in transcription initiation.

Explanation

The correct answer involves the interaction between viral and cellular factors during the initiation process, RNApolII recognition of the core promoter (TATA and +1), formation of the initiation complex leading to nascent RNA synthesis by RNApol I. The incorrect answers provide plausible but incorrect information related to various factors and processes in transcription initiation.

4. Draw the structure of a PolII promoter.

3.

1

4.

2.

The PolII promoter structure consists of various elements that regulate gene transcription, including TATA box, transcription start site, and regulatory sequences. It is essential for initiating the transcription of genes by RNA polymerase II.

Explanation

The PolII promoter structure consists of various elements that regulate gene transcription, including TATA box, transcription start site, and regulatory sequences. It is essential for initiating the transcription of genes by RNA polymerase II.

5. What is the function of PolII promoter structure?

2. have no role in determining transcription specificity and efficiency

3. are solely responsible for DNA replication during cell division

1. use specific cellular proteins that bind to help with transcription2. use cellular proteins that recognize this region and their availability in cells determine permissively3. enhance transcription of very fist genes transcribed i.e enhancer-binding proteins bridge DNA to initiate transcription

1. control gene expression only in bacteria by regulating RNAP

The PolII promoter structure plays a crucial role in transcription by utilizing cellular proteins, recognizing specific regions, and enhancing gene transcription through enhancer-binding proteins. The incorrect answers provided do not accurately describe the function of PolII promoter structure.

Explanation

The PolII promoter structure plays a crucial role in transcription by utilizing cellular proteins, recognizing specific regions, and enhancing gene transcription through enhancer-binding proteins. The incorrect answers provided do not accurately describe the function of PolII promoter structure.

6. (Transcription factors)1. 2.3.4.5..

5..

1. contain regions that specify binding to each other, DNA sites and cellular transcription2. Viral transcription factors are configured the same ways as cellular transcription factors3. DNA binding is specific protein-protein interaction4. cellular transcription factors is often not sufficient, need viral transcription factors

2. Viral transcription factors are configured the same ways as cellular transcription factors

3. DNA binding is specific protein-protein interaction

Transcription factors play a crucial role in gene expression by binding to specific DNA sites and regulating the transcription process. While viral transcription factors may share similarities with cellular transcription factors, they are not configured in exactly the same way. Additionally, DNA binding by transcription factors does not solely involve protein-protein interactions, as DNA sites are specifically targeted. It is important to note that cellular transcription factors may not always be sufficient for viral transcription, which may require additional viral transcription factors.

Explanation

Transcription factors play a crucial role in gene expression by binding to specific DNA sites and regulating the transcription process. While viral transcription factors may share similarities with cellular transcription factors, they are not configured in exactly the same way. Additionally, DNA binding by transcription factors does not solely involve protein-protein interactions, as DNA sites are specifically targeted. It is important to note that cellular transcription factors may not always be sufficient for viral transcription, which may require additional viral transcription factors.

7. What is in-vitro transcription?

A. reconstituted cellular transcription with purified transcription factors in a tube (with or without viral regulatory proteins present or with altered promoter/templates

B. process of cloning genes in a laboratory setting using restriction enzymes.

A. synthesis of DNA in a test tube without the involvement of cellular machinery.

C. conversion of RNA into DNA in a controlled environment outside of a living cell.

In-vitro transcription refers to the reconstitution of cellular transcription using purified transcription factors in a tube, with the flexibility of adding viral regulatory proteins or altering promoter/templates.

Explanation

In-vitro transcription refers to the reconstitution of cellular transcription using purified transcription factors in a tube, with the flexibility of adding viral regulatory proteins or altering promoter/templates.

8. Methods to assay gene expression: 1. Direct measurement of viral mRNA synthesis-

D. nuclear run-on

C. primer extension

B. RT-pcr

A. northern blotb. RT-pcrc. primer extensiond. nuclear run-one. microarray analysis

9. What are the two common methods used to measure protein-DNA interactions?

1. Western blot and ELISA

2. PCR amplification and DNA sequencing

3. Southern blot and Northern blot

1. gel shift and DNAse footprinting2. ChiP (chromatin Immunoprecipitation

The correct methods for measuring protein-DNA interactions are gel shift and DNAse footprinting for in-vitro studies, and ChiP (chromatin Immunoprecipitation) for in-vivo studies.

Explanation

The correct methods for measuring protein-DNA interactions are gel shift and DNAse footprinting for in-vitro studies, and ChiP (chromatin Immunoprecipitation) for in-vivo studies.

10. Which of the following DNA viruses uses its own unique regulatory signals in promoter and initiates transcription on the virion structure before complete uncoating?

1. Pox virus: very large and replicates in cytoplasm; contains own unique regulatory signals in promoter;transcription is initiated on the virion structure before complete uncoating and mediated by vRNApol

Herpes virus: small-sized virus that replicates in the nucleus; transcription is initiated after complete uncoating.

Adenovirus: medium-sized virus that replicates in the nucleus; transcription is initiated after viral DNA enters the nucleus.

Hepatitis B virus: small-sized virus that replicates in the nucleus; transcription is initiated in the cytoplasm after viral DNA is converted to cccDNA.

11. What are the 4 modes of transcription for DNA viruses?

Non-retroviruses: do not integrate into cellular genome and express viral regulatory proteins.

Papillomaviruses: do not integrate into cellular genome and do not express viral regulatory proteins.

Adenoviruses: do not integrate into cellular genome and express viral regulatory proteins.

Retroviruses: integrates into cellular genome and do not express viral regulatory proteins.
Lentiviruses: "DO" express regulatory proteins because it is treated like any cellular gene when transcribed.
A. 1. complicated promoter/enhancer region (contains many binding sites for cellular factors)2. availability of factors in cell3. physical state of chromatin in the integration region

The 4 modes of transcription for DNA viruses involve different factors that influence the transcription process. Retroviruses integrate into the cellular genome and do not express viral regulatory proteins; Lentiviruses, however, do express regulatory proteins. The correct answer focuses on the complicated promoter/enhancer region, availability of factors in the cell, and the physical state of chromatin in the integration region.

Explanation

The 4 modes of transcription for DNA viruses involve different factors that influence the transcription process. Retroviruses integrate into the cellular genome and do not express viral regulatory proteins; Lentiviruses, however, do express regulatory proteins. The correct answer focuses on the complicated promoter/enhancer region, availability of factors in the cell, and the physical state of chromatin in the integration region.

12. What is the transcription mode of DNA viruses known as cascade regulation?

Random transcription

Single-strand transcription

Most viruses are expressed in a cascade effect. First genes expressed are cellular promoters that have complicated promoter enhancer regions. Proteins products of these genes are regulatory proteins that activates transcription of alter genes. DNA replication to increase number of templates or change physical state of template to activate late viral genes.

Bidirectional transcription

Cascade regulation is a mode of transcription where viral genes are expressed in a cascading effect, starting with cellular promoters that have complex regulatory regions. This process involves activating regulatory proteins that then trigger the transcription of other genes, leading to DNA replication and activation of late viral genes.

Explanation

Cascade regulation is a mode of transcription where viral genes are expressed in a cascading effect, starting with cellular promoters that have complex regulatory regions. This process involves activating regulatory proteins that then trigger the transcription of other genes, leading to DNA replication and activation of late viral genes.

13. What are the 4 modes of transcription of DNA viruses?

Some viruses induce cellular pathways by specific interaction with viral surface proteins with receptors on cell surface. This activates kinase cascade inside cell resulting in altered activity or abundance of cellular transcription factors. Then activates vgene transcription.

Direct binding of viral RNA polymerase to host cell DNA for transcription.

Initiation of transcription by viral RNA polymerase without the involvement of host cell machinery.

Viral DNA being transcribed directly into proteins without any intermediate steps.

Transcription of DNA viruses involves various mechanisms, where some viruses interact with cellular pathways to alter transcription factors and gene expression.

Explanation

Transcription of DNA viruses involves various mechanisms, where some viruses interact with cellular pathways to alter transcription factors and gene expression.

14. HIV transcription involves 2 steps: 1. HIV enhancer and 2. TAT. What are the functions of these steps?

1. activity is dependent on availability of cellular proteins. Reason why HIV grows in activated T cells and monocytes lineage cells. (NF-kb)
2. increase transcription from LTR creating positive regulatory loop. a. binds to structure at 5'end of nascent RNA called TAR b. has modular structure like many transcription factors.

1. TAT functions as a repressor of HIV transcription. 2. HIV enhancer activates the host immune response to eliminate the virus.

1. TAT is a structural protein of the HIV virus. 2. HIV enhancer is responsible for viral protein synthesis.

1. HIV enhancer is responsible for viral integration into host genome. 2. TAT is involved in viral packaging and assembly.

The correct answers highlight the roles of HIV enhancer and TAT in the transcription process of HIV. Option 1 incorrectly states the function of TAT, while Option 2 and 3 provide inaccurate information about the roles of HIV enhancer and TAT respectively.

Explanation

The correct answers highlight the roles of HIV enhancer and TAT in the transcription process of HIV. Option 1 incorrectly states the function of TAT, while Option 2 and 3 provide inaccurate information about the roles of HIV enhancer and TAT respectively.

15. HIV transcription effects: initiates due to enhancer to express TAT only because more often than not Pol falls off.

B. increase phosphorylation of CTD of RNApol II decrease processivity causing truncated RNA

A. increase TAT binds to TAR and inhibits recruitment of cellular kinases

Initiates due to enhancer to express TAT only because more often than not Pol falls off.a. decrease TAT binds to TAR and recruits cellular kinasesb. decrease phosphorylation of CTD of RNApol II increase processivity causing full length RNA

C. decrease TAT binds to TAR and hinders recruitment of cellular kinases

Transcription of HIV is a complex process where the interplay of factors like enhancers, TAT, Pol, and phosphorylation affect the outcome. HIV transcription is initiated by an enhancer leading to the expression of TAT. In cases where Pol falls off, the process can be disrupted, impacting the binding of TAT to TAR, recruitment of cellular kinases, and the processivity of RNApol II.

Explanation

Transcription of HIV is a complex process where the interplay of factors like enhancers, TAT, Pol, and phosphorylation affect the outcome. HIV transcription is initiated by an enhancer leading to the expression of TAT. In cases where Pol falls off, the process can be disrupted, impacting the binding of TAT to TAR, recruitment of cellular kinases, and the processivity of RNApol II.

16. Adenovirus transcription: 1.2.3..

E1 genes are transcribed first followed by E2 genes.

1. first genes to be transcribed are E! region via strong enhancer region2. Remaining E transcription units are activated by E1A3. E1A and late genes activates late transcritption unit (cascade regulation)

E1A and E1B genes are transcribed simultaneously in the early stage.

Transcription of adenovirus does not involve any enhancer regions.

The correct sequence of events in adenovirus transcription involves the E1 region being transcribed first, followed by activation of remaining E transcription units by E1A, and then activation of late transcription unit by E1A and late genes for cascade regulation.

Explanation

The correct sequence of events in adenovirus transcription involves the E1 region being transcribed first, followed by activation of remaining E transcription units by E1A, and then activation of late transcription unit by E1A and late genes for cascade regulation.

17. What is the function of E1A in Adenovirus transcription?

2. Inhibits the binding of E2F to RB, leading to transcriptional repression

1. binds to RB (RB normally interacts with E2F and inhibits its ability to stimulate transcription2. E1A-RB binding allows e2F to act as an activator for transcription resulting in activation of E2 region of Adenovirus which encodes vDNA proteins and S-phase genes in cells, which encodes cellular DNA synthetic protein. (Both needed)3. Interacts with p300 which binds to histone acetylate and targets it to promoters resulting in acetylate histones and enhancer activation. (Binding of E1A to p300 results in enhancer dependent transcription repression.

3. Interacts with p53 to induce apoptosis in infected cells

4. Enhances the host cell's immune response to Adenovirus infection

E1A binds to RB, releasing E2F to activate transcription of Adenovirus genes. This process is essential for viral replication and production of viral proteins.

Explanation

E1A binds to RB, releasing E2F to activate transcription of Adenovirus genes. This process is essential for viral replication and production of viral proteins.

18. What is the structure of HSV in terms of transcription?

Linear genome with introns allowing for splicing of genes.

Circular genome with multiple genes sharing a single promoter.

Linear genome with each gene having its own promoter. No introns which inhibits splicing and promoter transport of unspliced genes.

Circular genome with no promoters for gene transcription.

HSV has a linear genome with each gene having its own promoter, allowing for individual regulation of gene expression. The lack of introns in HSV genes prevents splicing and allows for the direct transcription of unspliced genes.

Explanation

HSV has a linear genome with each gene having its own promoter, allowing for individual regulation of gene expression. The lack of introns in HSV genes prevents splicing and allows for the direct transcription of unspliced genes.

19. Activation of IE promoters by VP16:HSV doesn't rely on strong cellular enhancers, it brings its own activator to stimulate IE gene expression. VP16-

Is a late protein , packaged with mature virion. Performs a structural role as well. Located in the tegument region of virion (between envelope and nucleocapsid)a. forms a complex with Oct 1 and HCF cellular proteins then binds to TATA elements in the promoters of IE genes (IE-immediate/early)b. VP16 activation domain interacts with TBP, TFIIB and TAF to enhance recruitment of RNApol II to promoter. (TBP-trans. binds proteins, TFIIB trans. factor 2B + TAF =trans. act. factor

B. VP16 activation domain interacts with TBP, TFIIB and TAF to enhance recruitment of RNApol II to promoter. (TBP-transcription factor binds proteins, TFIIB transcription factor 2B + TAF = transcription activation factor)

D. VP16 promotes the proliferation of host cells rather than the activation of specific viral genes.

C. VP16 is involved in viral replication and assembly, not in gene transcription regulation.

VP16 is a late protein packaged with mature virion. It performs a structural role and is located in the tegument region of the virion, between the envelope and nucleocapsid.

Explanation

VP16 is a late protein packaged with mature virion. It performs a structural role and is located in the tegument region of the virion, between the envelope and nucleocapsid.

20. Activation of IE promoters by VP16:HSV doesn't rely on strong cellular enhancers, it brings its own activator to stimulate IE gene expression. Which of the following statements about VP16 and ICP4 is true during the lytic cycle of the virus?

A. activates the remainder of HSV genesb. binds TFIID (earlier stage than VP16) to TAT box and mediate other transcription factors (forming an activated transcription complex)c. multimerizes on DNA increasing the affinity for genome.

*VP16 and ICP4 are mainly during the lytic cycle of the virus.

B. binds TFIID (earlier stage than VP16) to TAT box and mediate other transcription factors (forming an activated transcription complex)

C. multimerizes on DNA increasing the affinity for genome

During the lytic cycle of the virus, VP16 mainly activates the remainder of HSV genes. While ICP4 may have different functions mentioned, it is VP16's activation role that is emphasized in this context.

Explanation

During the lytic cycle of the virus, VP16 mainly activates the remainder of HSV genes. While ICP4 may have different functions mentioned, it is VP16's activation role that is emphasized in this context.

21. Activation of IE promoters by VP16:HSV doesn't rely on strong cellular enhancers, it brings its own activator to stimulate IE gene expression.

1. Cellular enhancers are essential for activating IE promoters by VP16:HSV.

1. Product of IE gene ring finger position2. general activator in transient assays3. required for efficient viral growth and reactivation from latency4. reactivation of quiescent genome5. ubiquitin E3 ligase affects the abundance of target proteins6. dissociates repressive nuclear structures and inhibits HDAC

2. VP16:HSV relies on external cellular factors to stimulate IE gene expression.

3. The activation of IE promoters by VP16:HSV is not significant for viral growth and latency reactivation.

22. What is the role of Ori in DNA replication?

B) Ori is a protein that assists in DNA repair during replication.

1. a) Ori-small DNA sequence which is the site where replication begins b) replication proteins has one gene that is required for the proteins to bind to ORi
2. Plasmid- clone small pieces and see if Ori is tart of replication3. won't replicate because Ori isn't integrated and no replication proteins a. distriguishes replication and unreplicated DNA how: bacterial DNA is methylated, methylated in human cells in a way to regulate gene expression (both strands). Human cells do not methylate at A-residue (but the cytosine residue)

C) Ori is a molecule that regulates cell division during replication.

A) Ori is a type of enzyme that breaks down DNA strands during replication.

Ori is a specific DNA sequence where DNA replication begins, and replication proteins are required to bind to Ori for the replication process to initiate. It is not involved in breaking down DNA strands, DNA repair, or cell division regulation during replication.

Explanation

Ori is a specific DNA sequence where DNA replication begins, and replication proteins are required to bind to Ori for the replication process to initiate. It is not involved in breaking down DNA strands, DNA repair, or cell division regulation during replication.

23. (Replication of DNA)DNApol:1. alpha, sigma, epsilon2. beta.

3. are for translation and have high fidelity and processivity

4. are for recombination and have high fidelity and processivity

2. are for transcription and have low fidelity and processivity

1. are for replication and contains high fidelity and sigma & epsilon both have high processivity2. DNA repair with low fidelity and processivity

The correct answer explains the roles of alpha, sigma, and epsilon in DNA replication, highlighting their high fidelity and processivity. The incorrect answers provide different functions of DNApol that do not align with its actual role.

Explanation

The correct answer explains the roles of alpha, sigma, and epsilon in DNA replication, highlighting their high fidelity and processivity. The incorrect answers provide different functions of DNApol that do not align with its actual role.

24. What are the two types of DNA replication?

1. Unidirectional replication, recognized 5' OH group of primer and RNA primers

1. bidirectional replication, recognized 3' OH group of primer and RNA primers
2. nonRNA primer and replicate on single strand

3. Non-bidirectional replication, recognized 3' OH group of primer and DNA primers

2. Bidirectional replication, recognized 5' OH group of primer and DNA primers

The correct types of replication are bidirectional replication, where the 3' OH group of primer and RNA primers are recognized, and non-RNA primer replication where replication occurs on a single strand.

Explanation

The correct types of replication are bidirectional replication, where the 3' OH group of primer and RNA primers are recognized, and non-RNA primer replication where replication occurs on a single strand.

25. What are the 3 modes of replication according to SV40 replication?

1. recognizes the origin, major early gene. binds to core origin to form a hexamer that encases the DNA.
a. binds to an AT rich palidrome & early palidrome from DNAse I
b. cause conformational change & stable unwinding using ssbp-A (Rp-A)
c. helicase activity with RP-A & topoisomerase I unwinds the origin.

4. forms a dimer with topoisomerase II to stabilize the replication fork.

3. utilizes RNA polymerase for unwinding the DNA strands during replication.

2. initiates replication by binding to a GC rich palindrome in the DNA.

In SV40 replication, the correct mode involves recognizing the origin, binding to the core origin, and forming a hexamer to encase the DNA. Options 2, 3, and 4 do not accurately describe the modes of replication according to SV40 replication.

Explanation

In SV40 replication, the correct mode involves recognizing the origin, binding to the core origin, and forming a hexamer to encase the DNA. Options 2, 3, and 4 do not accurately describe the modes of replication according to SV40 replication.

26. What are the 3 modes of replication in SV40 replication?

A. circular template is replicated by unwinding parental strand with a overwound region and the Topoisomerase I or II (one strand cleavage) causes the relaxing of the supercoil.
b. separation of the DNA replication product by Topoisomerase II (cleaves both strands) and relaxes the supercoling.

F. The replication process in SV40 involves the synthesis of new DNA strands without unwinding the parental strands.

E. Replication of DNA occurs without the involvement of any topoisomerase enzymes.

C. Linear template is replicated by winding parental strands with a underwound region and Topoisomerase I causes the formation of supercoil. d. DNA replication product is separated by Topoisomerase I and increases the supercoiling.