Biochemistry final

What do we mean by the modular structure of transcription factors?

The modular structure of transcription factors refers to their organization into distinct functional units or domains. Domains like the DNA binding domain, activation domain, or the flexible protein domain can be interchanged between different transcription factors to create hybrid proteins with new functional properties.

What are common DNA-binding motifs discussed in class

Helix-turn-helix

Homeodomain

Zinc-finger

Leucine Zipper

Basic helix-loop-helix

For transcription factors that form heterodimers, explain how partner choice can change the function of the transcription factor.

Partner choice is a mechanism by which transcription factors fine-tune their function, allowing for precise regulation of gene expression and contributing to cellular diversity and adaptation

Why are zinc finger motifs useful for developing zinc finger nucleases?

They allow for specific and targeted DNA cleavage. Each zinc finger motif can be engineered to recognize a specific 3 base pair sequence, and multiple zinc fingers can be linked together to recognize longer DNA sequences (up to 18 bp)

What role does the leucine zipper play in the bZIP family of transcription factors?

It facilitating homo- and heterodimerization. This dimerization is essential for the bZIP proteins to bind to DNA as dimers

What is the role of zinc in zinc-finger proteins?

In zinc-finger proteins, zinc ions are crucial for stabilizing the protein's structure, particularly the "finger" motif, and enabling it to interact with other molecules, including DNA, RNA, and other proteins

What important class of transcription factors possess the C4 zinc-finger motif?

Nuclear receptors

What strategies have evolved to regulate transcription factor activity? Be familiar with each and with the examples discussed in class.

Homeobox genes

Phosphorylation

Release by an inhibitor

Protein degradation

Ligand activation

What happens when homebox domain transcription factors are incorrectly expressed?

HOX genes play a vital role in specifying the identity of body structures and segments during embryogenesis

What extracellular signals lead to phosphorylation of cAMP response element binding (CREB) protein? Why does phosphorylation increase transcription by CREB?

Extracellular signals like growth factors, hormones, and nerve impulses can lead to CREB phosphorylation. Phosphorylation of CREB, primarily at serine 133, enhances its ability to activate transcription by facilitating its interaction with co-activators like CBP/p300

How does phosphorylation and dephosphorylation of NFAT affect its localization? What structural changes allow this?

Phosphorylation maintains NFAT in the cytoplasm, while dephosphorylation by calcineurin allows it to translocate to the nucleus. Phosphorylation in the cytoplasm masks the NLS and promotes NES exposure. Dephosphorylation by calcineurin induces a conformational change that exposes the NLS and masks the NES, leading to nuclear import and transcriptional activation

Briefly describe the Keap1-Nrf2 pathway of regulation. What component is the target of proteosomal degradation?

Function: This pathway primarily serves as the body's principal defense against oxidative and electrophilic stresses, protecting cells from damage caused by reactive oxygen species (ROS) and electrophiles.

Mechanism:

Under normal conditions, Nrf2, a transcription factor, is kept at low levels in the cytoplasm through its interaction with Keap1.

Keap1 functions as a component of an E3 ubiquitin ligase, which tags Nrf2 for ubiquitination and subsequent degradation by the proteasome.

Under stress conditions (e.g., oxidative stress), stress molecules modify specific cysteine residues within Keap1.

This modification leads to a conformational change in Keap1, inhibiting the ubiquitination and degradation of Nrf2.

Nrf2 accumulates in the cell, translocates to the nucleus, and activates the transcription of cytoprotective genes encoding antioxidant enzymes and other proteins.

Briefly describe the NFκB-IκBα Pathway of regulation. What component is the target of proteosomal degradation?

Function: This pathway is a major regulator of the inflammatory response, controlling the expression of genes involved in inflammation, immunity, and cell survival.

Mechanism:

NF-κB, a transcription factor, is normally kept inactive in the cytoplasm by binding to its inhibitor protein, IκBα.

Upon receiving an inflammatory stimulus (e.g., cytokines), the IKK complex is activated, which phosphorylates IκBα at specific serine residues.

Phosphorylation of IκBα triggers its ubiquitination by an E3 ubiquitin ligase complex, leading to its subsequent degradation by the proteasome.

With IκBα degraded, NF-κB is free to translocate to the nucleus, where it activates the transcription of target genes, including those encoding inflammatory cytokines and other mediators

Briefly describe the three mechanisms of transcription factor regulation involved in the unfolded protein response.

1. Translational: ATF4
2. Post-transcriptional: XBP-1
3. Post-translational: ATF6

Under normoxic conditions, what happens to HIF-1? What happens under hypoxic conditions?

Under normoxic conditions, HIF-1α is rapidly hydroxylated, marking it for ubiquitination and degradation by the proteasome. Under hypoxic conditions, HIF-1α is stabilized, translocates to the nucleus, and forms a complex with HIF-1β, activating transcription of target genes involved in cell proliferation, glycolysis, and other metabolic pathways

What are the properties of molecules that activate nuclear receptors?

Small hydrophobic molecules capable to diffuse freely through plasma and nuclear membranes

What are the roles of the P-box and D-box in the DNA binding domain of nuclear receptors?

P box residues make base-specific contacts with DNA

D box residues involved in dimerization and half site spacing

What are the four classes of nuclear receptors? What is an example of each?

Steroid receptors (Glucocorticoid), RXR heterodimers (ecdysone), Dimeric orphan receptors (HNF-4), and monomeric orphan receptors (NGFI-B).

Which nuclear receptor class binds to inverted repeats? Which classes bind direct repeats?

Homodimeric receptors binds to inverted repeats (Class 1 and 3)

Heterodimeric receptors binds to direct repeats (Class 2 and 4)

How is specificity of DNA binding by class II nuclear receptors determined?

Primarily by variable length of spacer nucleotide sequence.

In the absence of ligand, what kind of complex do class II nuclear receptors interact with and what effect does it have on transcription?

Corepressor protein, it leads to the repression of transcription.

In the presence of ligand, what kind of complex do class II nuclear receptors interact with and what effect does it have on transcription?

Coactivator proteins, generally activates transcription.

Which nuclear receptors had their binding sites expanded in the human genome by Alu-S SINEs? 

Retinoic acid and thyroid hormone response elements.

What are four functional effects of RNA editing?

Non-coding editing, exon editing, 3’ UTR/miRNA editing, and intron editing.

What are two major forms of editing?

A to I and C to U

Briefly describe how RNA editing occurs in trypanosomes.

RNA editing in trypanosome mitochondria involves the insertion and deletion of uridines into and from the primary transcript to create a mature, translated mRNA

What enzyme performs C-to-U changes in mammals?

Cytidine deaminase

What enzyme performs A-to-I changes in mammals?

Adenosine deaminase

What type of RNA editing occurs in the Apolipoprotein B gene, in which tissue and what is the result?

C to U, in the intestinal tissue, it causes the production of the ApoB48 protein which functions to absorb lipids from the intestine.

What type of RNA editing occurs in the GluR2 gene and what is the functional impact of the change?

A to I. It makes Ca+2 impermeable to the receptors it controls (AMPA)

Extensive editing occurs in plastids and mitochondria of plants, what types of changes occur in RNAs because of the editing?

This process involves nucleotide substitutions, with the most common being cytosine C to uridine U conversions. RNA editing in plant organelles leads to alterations in RNA sequences that can influence protein structure and function, affect tRNA maturation, and play a role in regulating intron splicing. This post-transcriptional modification process is essential for maintaining the proper function of chloroplasts and mitochondria and plays a significant role in plant development and adaptation

ADAR1 has a Z-DNA binding motif, what is one possible function for this domain?

Thought to be involved in binding to Z-RNA and reduces the development of lethal autoinflammation in mice by masking self-dsRNA from detection by antiviral dsRNA sensors.

What RNA editing enzymes play a role in viral immune defense and restrict retrotransposons activity?

ADAR1

Name several functional effects that RNA editing can play in health.

Protein Recoding

Alternative Splicing

RNA Stability

Promoting Cell Proliferation

Drug Resistance

What two types of disease could RNA editing contribute to as discussed in class?

Parkinson’s and Alzheimer diseases.

What are two potential biological advantages of RNA editing?

Regulatory control of gene expression at the post-transcriptional level

Potential of synthesizing two or more distinct products from a single gene

What is the primary way that siRNAs inhibit protein expression?

Endonucleolytic cleavage of mRNA

What is the primary way that miRNAs inhibit protein expression?

Translational repression; Degradation of mRNA endonucleolytic clevage of mRNA

Why are miRNAs able to target numerous different mRNAs whereas siRNAs are very specific to one target?

Their partial complementarity and ability to bind to multiple sites, whereas siRNAs, which require perfect complementarity, target a single mRNA

How do long non-coding RNAs effect gene expression in cells?

They can control the flux of genetic information, such as chromosome structure modulation, transcription, and splicing.

Understand the different methods for studying transcription factors

Chromatin Structure and accessibility methods
Electrophoretic mobility shift assay (gel shift)
Reporter gene assays
Computational searches for consensus sequences

Chromatin Structure and accessibility method

Provide insights into the complex organization of DNA and how this organization impacts gene regulation and other cellular processes

What three main properties of TEs allow them to have roles in gene regulation? 

TEs provide cis-regulatory sequences, encoding for regulatory RNAs, and insertion into genic regions and gene downregulation

Understand the difference between Class 1/Type 1 and Class 2/Type 2 transposable elements.

Type 1 are retro transposons, it includes LTR elements and non-LTR elements

Type 2 are DNA transposons

Understand how TEs can affect gene expression (e.g. exonization, hypereditiing and polyadenylation

Exoneration: expands the diversity of protein products generated from a single gene

Polyadenylation: The presence of a TE-derived PAS can lead to alternative polyadenylation, resulting in the production of mRNA variants with different 3'-untranslated regions (UTRs)

Explain how TEs may play a role in how HIV-1 infected individuals may control infection while others cannot. 

Low expression of some Krab-Zinc fingers leads to elevated basal expression of innate immune genes and restriction factors in EC individuals through an increase in the cis-regulatory activity of TEs serving as promoters or enhancers for these genes.

What TEs are implicated as risk factors for several major psychiatric conditions? What are the disorders?

Endogenous retroviruses, schizophrenia, bipolar disorder, and major depressive disorder

What kind of information is contained in viral genomes? 

Information that ensures replication of viral genomes, packaging of genomes into virons, and alters the structure and/or function of the host cell to a greater or lesser degree

Know the general life cycle of viruses. 

Attachment, penetration, and uncoiling

Gene expression and replication

Assembly and release

Describe the two types of nuclear DNA viruses concerning cellular location during replication. Know names of examples of each as discussed in class.

Nuclear DNA viruses: Parvoviruses, herpesviruses

Cytoplasmic DNA viruses: Poxviruses

Which DNA viruses depend heavily on host cell factors to express genes and replicate? 

Parvovirus

Which of the DNA viruses uses histones to package its genome into nucleosomes? 

Polyoma viruses and papilloma viruses

Briefly describe how the ssDNA genome of parvovirus is converted to a dsDNA molecule by host enzymes?

Inverted terminal repeat sequences at each end of the viral genome fold back on themselves. The free 3’ OH group at the end of the virus single-stranded genome is used as a primer for the synthesis of the complementary DNA strand. Uses host cell enzymes

Explain the importance of the parvovirus NS1 protein in viral replication. 

NS1 acts as an origin recognition protein. It binds to double stranded virus DNA and allows host DNA polymerase to replicate many single stranded copies of viral DNA strand

For some viruses, gene expression is divided into early and late phases. What proteins are primarily synthesized during the early phase and late phase? 

Early: E1A, Iva2

Late: E2A, L4P

During polyomavirus replication what viral protein is needed for DNA replication? Where does it bind? How does it regulate early and late gene expression? What effect can it have on host cells?

Large T antigen is needed. It binds at the origin of replication. T antigen controls increased transcription from the late promoter and decreased transcription from the early promoter. It also interacts with host proteins.

Describe the polyomavirus genome structure and how it replicates. 

Small genome, codes for few proteins. Circular dsDNA genome with histones. DNA replication is bidirectional and uses the host machinery.

Which of the small DNA viruses discussed in lecture makes its own DNA polymerase?

Adenovirus

Briefly describe adenovirus replication. How does it differ from DNA replication observed in the host cell?

One strand is replicated at a time, releases single strand DNA which is then copied into a double-stranded DNA. TP-dC bound to the 5’ strand mimics dsDNA, allows DNA polymerase to begin synthesis. Adenovirus polymerase can bind the panhandle duplex, read the strand from the 3’ to 5’ and hence synthesis the complementary 5’to 3’ strand

What sort of hybrid molecule does adenovirus use as a primer for replication?

TP-dC is it’s primer.

What sort of structure does the displaced strand form during adenovirus replication? What region of this structure allows synthesis of the displaced strand?

Pan handle duplex. Duplex region allows synthesis

What large DNA virus discussed in class replicates in the nucleus?  

Herpes viruses

What host activities does herpes require? Not require? 

RNA polymerase, mRNA modification enzymes. DNA polymerase, DNA binding protein, thymidine kinase, or ribonucleotide reductase.

What herpesvirus encoded proteins might make good therapeutic targets to inhibit its infection? 

VP16

Does herpesvirus replication depend on host replication machinery? 

They are more independent, but do require some host machinery

What sort of genomic structure is generated during replication and how is it modified for packaging into virions? 

They are long concatemeric after replication but are cleaved into genome size lengths when packaged.

What type of host proteins are needed by herpesvirus during replication and production of virus? 

RNA polymerase, mRNA modification enzymes, repair proteins.

Where do poxviruses replicate? What proteins/machinery must they provide to do so? 

In the cytoplasm. The provide their own mRNA and DNA synthetic machinery

Briefly describe the structure of the poxvirus genome. Complex morphology

They are large, double stranded DNA viruses, larger than herpes.

Briefly describe the steps to replicate the poxvirus genome. 

Host cells helicase nicks one end of DNA duplex. Terminal repeats fold over as hydrogen bonds form between complementary bases on same strand, forming hairpin loops. The free 3’ end folded over on itself in the hairpin loop to make it appear double stranded acts as a primer for DNA synthesis. DNA polymerase can bind this 3’ end and synthesis complementary DNA to the 3’ to 5’ strand. DNA synthesis occurs around the other terminal loop, resulting in two genomes joined end to end.

What enzyme do RNA viruses provide to replicate their genome? The host does not provide it. 

RNA dependent RNA polymerase

Describe the four major types or classes of RNA viruses discussed in class. What is the basis of their classification? Provide one example of each class that we discussed in class (e.g. retrovirus, HIV1) 

Plus stranded: translation occurs upon infection; polio

Negative stranded: Need to copy into plus strand for translation; influenza

Double stranded: Need to make mRNA for translation; rotavirus

Retroviruses: reverse transcribes the genome into DNA and integrates into host genome; HIV

Which class of RNA virus has an infectious genome? 

Plus-stranded viruses

Which class of RNA virus has a DNA intermediate? 

Retroviruses

Which classes of RNA virus can have segmented genomes? 

Double stranded, negative stranded,

How do small RNA viruses increase the protein coding capacity of their genomes?

Overlapping open reading frames and polymerase stuttering

Describe two strategies RNA viruses use to encode more than one protein from a monocistronic RNA. 

Viruses makes primary transcripts which are processed by host splicing machinery to yield more than one monocistronic RNA. The virus makes multiple monocistronic mRNAs.

How can introducing only the poliovirus genome into a cell cause an infection? 

Genome translation yields a single polyprotein that is proteotically cleaved into replication proteins and capsid proteins. Genome replication by the viral RNA dependent RNA polymerase starts with synthesis of a negative strand that serves as a template for synthesis of new RNA molecules.

Picornaviruses like poliovirus lack a 5’-methylated cap structure, how are they translated? How does the virus interfere with host translation? 

It has a ribosome landing pad (IRES), the ribosomes bind without having to recognize the 5’ methylated cap. It interferes with host translation by shifting translation away from capped mRNAs to uncapped viral RNAs. A single polypeptide is produced and cleaved.

What protein does poliovirus require as a primer for replication? 

VPg

How do negative-strand RNA viruses protect their genome? 

The virion RNA polymerase can act on the viron RNA in it’s nucleocapsid form which protects it from ribonucleases. There is one monocistronic mRNA for each of the five virally coded proteins. They are capped. Methylated, and poly adenylated by enzymes packaged in the virion. All virion proteins are made at the same time.

Describe the transcription model for non-segmented, negative-stranded RNA viruses like measles virus. How does this affect the levels of the different transcripts that are made? 

Single-entry, stop-start transcription model. The polymerase encoded by the L gene generates a 5’-terminal cap and polyadenylates mRNAs by polymerase slippage. Gene start-stop sequences leads to termination and initiation of each mRNA. Gradient of expression.

Accumulation of what two proteins leads to a switch from transcription to replication for negative-stranded, non-segmented viruses? 

Viral RNA free N and P protein complexes.

The segmented, negative-stranded virus influenza has an unique way of adding a 5’-methylated end to its own mRNAs. How does it do this? 

A endonuclease packaged in the virus snips off the 5’ end of host capped, methylated mRNA about 13-15 bases from the 5’ end and uses this as a primer for viral mRNA synthesis. All flu mRNAs have a short stretch at the 5’ end which is derived from the host mRNA.

Describe the template used for negative-stranded virus (segmented or non-segmented) replication.

A full length positive strand RNA coated with nucleocapsid protein is used as a template to synthesize full-length negative strands which are coated with nucleocapsid proteins. This can be used as a template for replication..

What form of the rotavirus serves as a site for viral mRNA transcription? Slide 19

Double layered particles (DLPs)

Segmented virus genomes allow what process to occur? How is it an advantage to the virus?

Genetic exchange in a cell infected by more than one virus. Ex. Two different influenza viruses co-infect a cell. This reassortment is responsible for some of the major genetic shifts in the history of the influenza virus.

Briefly describe how retroviruses integrate into the host genome after entering the cell. 

RNA is copied by reverse transcriptase to minus send DNA. The polymerase is acting as an RNA-dependent DNA polymerase and it needs a primer, which is a tRNA incorporated into the virus particle from the previous host cell. RNA is displaced and degraded by a virus-encoded RNase H activity. Reverse transcriptase now acts as a DNA dependent DNA polymerase and copies the new DNA into a double strand DNA. The DNA circulizes and is integrated into the host cell DNA by virally encoded integrase to become a provirus. It is now synthesized by RNA polymerase into a long mRNA that is capped and polyadenylated. It is translated into GAG and POL polyproteins. It is also processed to other mRNAs for additional viral proteins.

How do retroviruses ensure the genome is replicated from the provirus with all the appropriate regulatory information? Do not describe the details outlined in the lecture, but summarize what happens during generation of the provirus. 

Both dsDNA have U3-R-U5 sequences, called LTR sequences. LTRs mediate the integration of the retroviral DNA into another region of the host genome.

What is the biological role for CRISPR in bacteria?

Provide an adaptive immune system against invading genetic elements. The system allows bacteria to remember past infections and mount a targeted defense, contributing to their survival and evolution in environments teeming with phages and other genetic parasites

Explain how how CRISPR/Cas9 functions to target an invading pathogen? What is the role of the tracrRNA, crRNA and Cas9.

crRNA and tracrRNA in type 2 CRISPR systems form a complex known as the guide RNA or gRNA. The crRNA within the gRNA is what matches up with the target sequence or protospacer after the PAM is found. Once the match is made, Cas9 will make a double stranded break.

How does CRISPR/Cas9 distinguish foreign DNA from host DNA?

Protospacer-adjacent motif (PAM) of the invader’s genome is the essential targeting component which distinguishes bacterial self from non-self-DNA.

Scientists have adopted CRISPR/Cas9 for targeted genome editing, how does the gRNA differ from that found in bacteria? 

While the natural bacterial CRISPR-Cas9 system uses a two-RNA guide (crRNA:tracrRNA duplex), the adapted system for genome editing commonly utilizes a single guide RNA (sgRNA) for increased simplicity, efficiency, and flexibility in manipulating the genome

Describe the various engineering options that one can use with the CRISPR/Cas9 system.

Genome engineering with Cas9 nuclease

Genome engineering by double nicking with paired Cas9 nickases

Localization with defective Cas9 nuclease.

Which engineering option enhances the production of homology direct repair?  

Genome engineering by double nicking with paired Cas9 nickases

BE4

Fusion of nCas9 with APOBEC1, can prevent the excision of uracil by endogenous uracil N-glycosylase enzymes

Electrophoretic mobility shift assay (gel shift)

Provides information about protein-DNA interactions by observing the shift in DNA mobility during gel electrophoresis when a protein binds to it

Reporter gene assays

Provide insights into gene regulation, particularly focusing on promoter activity and the effects of different factors on gene expression

Computational searches for consensus sequences

Provide valuable information about the similarities and functional significance of DNA, RNA, or protein sequences within a family or set

ABEs

Fusion of tRNA adenosine deaminase with evolved TadA domain