9.2

What is the main goal of Chapter 9.2?

To determine what proteins do by studying their sequences, expression, location, interactions, and effects when altered.

What are the three levels of protein function?

Phenotypic, cellular, and molecular function.

What is phenotypic function?

The effect of a protein on the whole organism, such as growth, development, or survival.

What is cellular function?

The protein’s role in cellular processes and its interactions with other cell components.

What is molecular function?

The precise biochemical activity of a protein, such as catalyzing a reaction or binding a ligand.

What is a transcriptome?

The complete collection of RNA transcripts present in a cell at a particular time.

What is a proteome?

The complete collection of proteins present in a cell at a particular time.

What is transcriptomics?

The study of RNA transcripts and how their levels change under different conditions.

What is proteomics?

The large-scale study of cellular proteins, including abundance and modifications.

What is comparative genomics?

Comparing DNA or protein sequences across organisms to predict gene and protein functions.

What is genome annotation?

Identifying genes and other features in a DNA sequence and assigning possible functions.

What does BLAST do?

Searches sequence databases for DNA or protein sequences similar to a query sequence.

How can sequence similarity suggest protein function?

A newly discovered protein may have a similar function to a previously studied protein with a related sequence.

What are orthologs?

Related genes in different species that descended from a common ancestral gene.

What are paralogs?

Related genes that arose through gene duplication, often within the same species.

What is synteny?

Conservation of gene order along corresponding chromosome regions in different species.

Why does conserved gene order help identify orthologs?

Matching neighboring genes provides extra evidence that two genes share an evolutionary origin.

How can a protein structural motif help predict function?

A known motif may suggest ATP binding, DNA binding, zinc binding, or another molecular activity.

Why are conserved active-site sequences useful?

They can reveal catalytic residues and suggest how an enzyme carries out its reaction.

Why does a protein’s location help reveal its function?

A protein must generally be present where and when the process it participates in occurs.

What is RNA-Seq used for?

Identifying and estimating the relative abundance of RNAs expressed in cells or tissues.

What are the basic steps of RNA-Seq?

Isolate RNA, fragment it, convert it to cDNA, sequence the DNA, and analyze the reads.

Why is RNA converted to cDNA for RNA-Seq?

DNA sequencing methods can then be used to identify and quantify the original RNAs.

What do more RNA-Seq reads for a transcript generally indicate?

That the RNA transcript was more abundant in the sample, after appropriate analysis.

What is single-cell RNA-Seq?

RNA-Seq performed on individual cells to compare expression between different cell types or states.

How can RNA-Seq help study cancer?

It reveals differences in gene expression among normal and tumor cells or tumor cell populations.

Does abundant mRNA always mean abundant protein?

No. RNA levels do not perfectly predict protein abundance because translation and protein turnover vary.

What does mass spectrometry reveal about the proteome?

Which proteins are present, their relative amounts, and sometimes their modifications.

RNA-Seq versus mass spectrometry: what does each measure?

RNA-Seq measures RNA transcripts; mass spectrometry analyzes proteins.

What is a fusion protein?

A protein made by joining coding sequences so that two protein parts are produced as one polypeptide.

What is GFP?

Green fluorescent protein, a fluorescent marker used to visualize protein location and movement.

How is GFP used to locate a target protein?

Fuse the GFP gene to the target gene so the resulting protein fluoresces in cells.

What is the original biological source of GFP described in the chapter?

The jellyfish Aequorea victoria.

What is a major advantage of GFP fusions?

They can reveal protein location and movement in living cells.

What is a limitation of GFP fusion proteins?

The GFP tag may alter protein folding, activity, or expression.

What is immunofluorescence?

Using fluorescently labeled antibodies to visualize where a specific protein is located.

What is an epitope?

A specific region of a protein recognized and bound by an antibody.

What is the role of the primary antibody in indirect immunofluorescence?

It binds specifically to the protein of interest.

What is the role of the fluorescent secondary antibody?

It binds the primary antibody and provides the detectable fluorescent signal.

GFP versus immunofluorescence: key difference?

GFP uses a fluorescent fusion protein and can track living cells; immunofluorescence uses antibodies, usually in fixed cells.

What does biotin-streptavidin binding help with in immunofluorescence?

It provides a strong, specific connection that can help label and detect antibody-bound proteins.

Why study protein-protein interactions?

A protein’s binding partners can reveal the cellular processes and complexes in which it functions.

What is immunoprecipitation?

Using an antibody to isolate a target protein from a cell extract, sometimes with associated proteins.

What is co-immunoprecipitation (co-IP)?

Pulling down a target protein and detecting proteins associated with it.

What is an affinity tag?

An attached peptide or protein that allows a target protein to bind a specific purification material.

What is tandem affinity purification (TAP)?

Two consecutive affinity purification steps using two tags to isolate a protein complex.

Why use two steps in TAP?

To remove weakly bound contaminants and reduce false-positive protein interactions.

What are the two tag components in the chapter’s TAP example?

Protein A and a calmodulin-binding peptide, separated by a TEV protease cleavage site.

What does protein A bind during TAP?

The Fc region of immunoglobulin G (IgG) on the first affinity column.

What does TEV protease do in TAP?

Cleaves a specific site to release the tagged protein complex from the first purification step.

What does yeast two-hybrid analysis test?

Whether two proteins interact with one another in a yeast cell.

Which two Gal4 parts are separated in yeast two-hybrid analysis?

The DNA-binding domain and the transcription activation domain.

How does a positive yeast two-hybrid result occur?

Interacting test proteins bring the Gal4 domains together and activate a reporter gene.

What is a reporter gene?

A gene whose detectable product indicates that a specific event, such as protein interaction, occurred.

Why might yeast two-hybrid give a false positive?

An apparent interaction can be indirect or involve other proteins in a larger complex.

How can deleting a gene help determine protein function?

Scientists observe what changes when the cell can no longer make a functional version of that protein.

What is CRISPR originally used for in bacteria?

Defense against bacteriophages by recognizing and destroying matching viral DNA.

What are CRISPR spacer sequences?

DNA segments derived from earlier viral encounters that help bacteria recognize related viruses.

What does Cas9 do?

Acts as a programmable nuclease that can cut both strands of target DNA.

What does single-guide RNA (sgRNA) do?

Directs Cas9 to a complementary target DNA sequence.

What two RNA components are combined in an sgRNA?

A CRISPR RNA (crRNA) guide and a trans-activating CRISPR RNA (tracrRNA) scaffold.

Why can Cas9 cut both DNA strands?

It has two nuclease domains that cleave opposite DNA strands.

What is nonhomologous end joining (NHEJ)?

A DNA break-repair pathway that joins broken ends and often introduces small insertions or deletions.

How can NHEJ produce a gene knockout?

Repair errors can disrupt the coding sequence so a functional protein is no longer produced.

How can a specific DNA change be introduced after CRISPR cutting?

Provide a donor DNA template with the desired change for template-directed repair.

What is a Cas9 nickase?

A modified Cas9 that cuts only one DNA strand rather than both.

What is an off-target effect in CRISPR?

An unintended cut or change at a DNA sequence other than the intended target.

What is a gene drive?

A genetic system that biases inheritance so a genetic change can spread through a population more often than normal Mendelian inheritance predicts.

What is the X-shredder gene-drive example meant to accomplish?

Bias mosquito offspring toward males and potentially reduce a target population over generations.

What is an important concern about releasing gene drives?

They may spread unexpectedly or have unintended ecological consequences.

What is high-throughput genetic screening?

Testing the effects of many different gene perturbations across a large population of cells.

What is a gene perturbation?

An experimental change that inactivates, activates, or otherwise alters a gene’s expression or function.

How is a CRISPR sgRNA library used in genetic screening?

Different cells receive different guides so many genes can be tested in parallel.

Why can sgRNA sequences act like barcodes?

Each guide sequence identifies the gene targeted in a particular cell or group of cells.

What happens after cells in a genetic screen are exposed to a stress?

Cells with harmful gene perturbations may disappear, while cells with beneficial changes may become enriched.

How are enriched or depleted sgRNAs identified?

By deep DNA sequencing of guide sequences before and after the selection.

What is dead Cas9 (dCas9)?

A modified Cas9 that binds guide-directed DNA but lacks DNA-cutting activity.

What is CRISPR interference (CRISPRi)?

Using dCas9, often with a repressor, to reduce transcription of a target gene.

What is CRISPR activation (CRISPRa)?

Using dCas9 fused to an activator to increase transcription of a target gene.

Regular Cas9 versus dCas9: main difference?

Regular Cas9 cuts DNA; dCas9 binds DNA without cutting and can be used to regulate transcription.

Which technique should you use to find similar genes in different species?

Comparative genomics with sequence searches such as BLAST.

Which technique should you use to locate a protein in a living cell?

GFP fusion microscopy.

Which technique should you use to test whether two proteins interact?

Yeast two-hybrid analysis; co-IP or TAP can also help study associated proteins.

Which technique should you use to test the effect of losing a gene?

CRISPR/Cas9 gene disruption followed by examination of the resulting phenotype.