Cell and Molec Lecture 9A

Why can cells in the same organism have very different structures and functions?

Because different cell types express different sets of genes, causing them to produce different RNAs and proteins at different concentrations.

How does gene expression differ between a neuron and a liver cell?

A neuron expresses genes needed for structures and functions such as dendrites, axons, sensory integration, and signaling, while a liver cell expresses genes involved in detoxification, synthesis, metabolism, and resource storage.

What are the major structural features of a neuron?

It has a soma (cell body), multiple branching dendrites, and a long axon with branches called collaterals.

What is the function of neuronal dendrites?

They receive signals from other neurons and sensory cells.

What is the function of a neuron's axon and axon collaterals?

They transmit signals to other neurons and effector cells

What is the relationship between cell morphology/function and gene expression?

Differences in morphology and function are largely products of differences in gene expression between cell types.

What is GFP?

Green fluorescent protein, a reporter protein that produces green fluorescence and can be used to show where a gene is being expressed.

What is a reporter gene?

A gene whose expression produces an easily detectable signal, allowing researchers to determine where and when another gene or regulatory region is active.

Where was the GFP sequence placed in the zebrafish experiment?

Into the GATA-1 gene locus, under the control of the GATA-1 gene's promoter.

Was GFP expressed in every cell of the zebrafish embryo?

No, GFP expression was restricted to specific cells and tissues.

Do cells that do not express a particular gene lack that gene?

No, a cell can contain a gene without expressing it.

What is the important distinction between having a gene and expressing a gene?

Having a gene means its DNA sequence is present in the genome; expressing a gene means the cell is using that DNA information to produce RNA and potentially protein.

Which cells are an exception to the statement that human cells contain the same genetic information?

Mature erythrocytes (red blood cells) lack a nucleus and therefore do not contain nuclear DNA.

What major conclusion about differentiated cells was demonstrated by John Gurdon's experiment?

The nucleus of a mature differentiated cell still contains all the genetic information needed to produce a complete organism

What type of cells did Gurdon use as the source of nuclei?

Frog skin cells.

What did Gurdon do to the unfertilized frog egg?

He destroyed the egg's nucleus using UV light.

What is nuclear transfer?

A technique in which the nucleus from one cell is transferred into an egg cell whose original nucleus has been removed or destroyed.

28. Q: What happened after Gurdon transferred a skin-cell nucleus into the enucleated egg?

A normal embryo developed and eventually produced a tadpole.

What did Gurdon's experiment demonstrate?

A mature differentiated cell nucleus still contains the complete genetic information required to produce an entire organism.

If a mature skin cell can produce a whole organism through nuclear transfer, why doesn't a skin cell normally become an entire organism?

Its genome is not missing genes; rather, its gene expression program has been specialized/reprogrammed for its differentiated state.

What does "reprogrammed" mean in the context of Gurdon's experiment?

It means that the genetic information in a differentiated nucleus can be placed in an environment that causes its gene expression patterns to change, allowing development to begin.

What famous cloned mammal does Dr. Nelson mention?

Dolly the sheep

What type of experimental evidence can be used to determine which genes are being expressed?

RNA sequencing (RNA-seq)

What is RNA-seq?

A next-generation sequencing technique used to analyze RNA transcripts in a sample.

What does RNA-seq allow researchers to determine?

Which genes are being expressed, how strongly they are expressed, and how their transcripts are spliced.

What is the transcriptome?

The collection of RNA transcripts present in a cell or sample under particular conditions.

Why is RNA-seq considered a transcriptome-level technique?

Because it can analyze many or essentially all of the transcripts present in a sample rather than examining only one gene at a time.

What is the general process of RNA-seq?

RNA is extracted, mRNA can be purified, the RNA is fragmented, converted into cDNA, converted into double-stranded DNA copies, sequenced, and the resulting reads are aligned to the genome.

What is cDNA?

Complementary DNA, DNA produced from an RNA template.

What are sequencing "reads"?

The individual DNA sequence fragments generated during sequencing

What happens to RNA-seq reads after sequencing?

They can be aligned to the genome to determine where the corresponding transcripts originated.

Why is RNA-seq considered quantitative?

It can provide information about the relative abundance of transcripts, allowing researchers to estimate expression levels.

What additional information can RNA-seq provide besides gene expression level?

It can provide information about RNA splicing and which transcript variants are produced.

What two genes were compared in the RNA-seq example?

Beta-actin and tyrosine aminotransferase.

What is beta-actin?

A protein that is an important component of the actin cytoskeleton.

Why is beta-actin considered a housekeeping gene?

Because it performs an essential function needed by many or all cells, so its gene is generally expressed broadly.

How was beta-actin expressed across the cell types in the RNA-seq

It was expressed in all of the samples at relatively similar levels.

What type of reaction does tyrosine aminotransferase perform?

A transamination reaction.

Is tyrosine aminotransferase expressed equally in all cell types?

No, it was expressed primarily by liver cells.

What is the protein-level equivalent of transcriptome analysis?

Proteomics

What technique is a proteomics method?

Two-dimensional gel electrophoresis.

What is the purpose of 2D gel electrophoresis?

To separate and visualize proteins based on two different properties, allowing protein expression patterns to be compared between samples.

What is done to cells or tissue before 2D gel electrophoresis?

They are broken open to produce a protein lysate, and the proteins are denatured.

What is a protein lysate?

A liquid mixture containing proteins released from cells or tissues after the cells are broken open.

What is the first dimension of 2D gel electrophoresis based on?

The protein's isoelectric point (pI).

What is the isoelectric point?

The pH at which a protein has no net electrical charge.

What is the second dimension of 2D gel electrophoresis based on?

Molecular weight

What does transcriptional control regulate?

Whether and how efficiently a particular gene is transcribed into RNA.

What is an example of RNA processing control?

RNA splicing.

Why does mRNA localization matter?

Where an mRNA is transported within the cell can influence where and when its protein product is produced.

Why does mRNA half-life affect gene expression?

An mRNA that persists longer can generally be available for translation for a longer period, potentially producing more protein.

What does translational control regulate?

How efficiently an mRNA is used by ribosomes to produce protein.

How can gene expression be controlled at the protein level?

By modulating the activity of the protein after it has been produced.

What are the six levels of gene expression control in order from most to least used?

Transcription

RNA processing

mRNA export/localization

mRNA stability/half-life

Translation

Protein activity

What is a transcriptional regulator?

A broad term for a protein that affects gene expression.

What are transcription factors?

Proteins that bind directly to specific DNA sequences and influence gene expression.

What DNA sequences do transcription factors bind?

They bind specific cis-regulatory sequences.

What does "cis" mean in cis-regulatory sequence?

It means the regulatory sequence is located on the same chromosome/DNA molecule as the gene it regulates.

What is a cis-regulatory sequence?

A DNA sequence on the same DNA molecule as a gene that can be recognized by regulatory proteins and influence that gene's expression.

Why don't transcription factors need to open the DNA double helix?

They can recognize chemical groups exposed along the edges of DNA bases in the major and minor grooves.

What are the major and minor grooves of DNA?

They are grooves along the outside of the DNA double helix where the edges of the bases are exposed.

Which DNA groove is wider?

The major groove

What types of chemical interactions can transcription factors make with DNA?

Hydrogen bonds, hydrophobic interactions, and other noncovalent interactions with DNA.

What types of chemical groups can be exposed in the DNA grooves?

Groups involved in hydrogen bonding as well as hydrophobic groups such as methyl groups.

How does a transcription factor achieve strong and specific DNA binding?

By making many weak interactions simultaneously with DNA.

Where do many transcription factors make their contacts with DNA?

Mostly in the major groove, although some contacts can occur in the minor groove and with the sugar-phosphate backbone.

What determines whether a transcription factor can bind a particular DNA sequence?

The specific arrangement of DNA bases must provide the appropriate chemical groups for the transcription factor's amino acid side chains to interact with.

Why are DNA-binding motifs important to know?

They are common features of transcription factors and are important for understanding how transcription factors recognize DNA.

What are the major DNA-binding motifs?

Helix-turn-helix

Homeodomain

Leucine zipper

Beta-sheet DNA recognition

Zinc finger

Helix-loop-helix

What is a helix-turn-helix motif?

A DNA-binding structure consisting primarily of two alpha helices connected by a short turn/linker.

What is the recognition helix in a helix-turn-helix motif?

The helix that directly interacts with and recognizes DNA bases.

Which helix acts as the recognition helix in the helix-turn-helix motif?

The C-terminal helix.

Approximately how far apart are the recognition helices in the helix-turn-helix dimers and why is it important?

Approximately 3.4 nanometers and It allows the two recognition helices to fit into successive major grooves of the DNA double helix.

What is a homeodomain?

A DNA-binding domain found in transcription factors encoded by homeotic selector genes.

What do homeodomain proteins regulate?

They play important roles in development.

How many alpha helices make up the homeodomain structure?

Three

What holds the three helices of a homeodomain together?

Hydrophobic interactions

Which homeodomain helix acts as the recognition helix?

Helix 3

What is a leucine zipper?

A DNA-binding/dimerization motif consisting of two long alpha helices that form a dimer through hydrophobic interactions.

What type of protein interaction forms the leucine zipper dimer?

Hydrophobic interactions between the surfaces of the two alpha helices.

What is a coiled-coil?

A structure in which two or more alpha helices wrap around one another, commonly through hydrophobic interactions.

What role does the coiled-coil portion play in a leucine zipper?

It forms the dimerization interface between the two protein subunits.

What happens beyond the coiled-coil region of a leucine zipper?

The alpha helices extend outward, forming a Y-shaped structure that can interact with DNA

How does a leucine zipper interact with DNA?

Its extended helices bind to major grooves on opposite sides of the DNA double helix.

What important transcription factor is a leucine zipper protein?

AP1

What protein is the beta-sheet DNA-binding protein?

The E. coli MetJ protein.

What does MetJ regulate?

It represses genes involved in methionine production.

Does the MetJ protein function as a monomer or dimer?

Dimer

What is the DNA recognition region of the beta-sheet protein made of?

A two-stranded antiparallel beta-sheet.

What is a zinc finger?

A DNA-binding structural motif in which a zinc atom helps stabilize the protein structure, producing a finger-like shape.

How does a zinc finger interact with DNA?

A portion of the zinc finger, often an alpha helix, interacts with DNA bases in the major groove.

What is the role of the zinc atom in a zinc finger?

It stabilizes and holds the motif together.

What is a helix-loop-helix motif?

A DNA-binding/dimerization motif consisting of two alpha-helical regions connected by a flexible loop.

What connects the two helices in a helix-loop-helix protein?

A flexible linker/loop region.

Which helices interact with DNA in the helix-loop-helix structure?

The longer helices interact with the major grooves on opposite sides of the DNA.

How large of a DNA sequence can an individual transcriptional regulator monomer typically recognize?

6-8 bases

What is a sequence logo used to represent?

It illustrates the sequence preferences of a DNA-binding protein, showing which bases are preferred at each position.

Why is binding to only a 6-base sequence potentially problematic in a large genome?

A 6-base sequence is likely to occur many times by chance, so the transcription factor could potentially bind many incorrect locations.

Why do many transcriptional regulators dimerize?

It increases their DNA-binding affinity and specificity.

How does dimerization increase DNA-binding specificity?

Two protein subunits can recognize a longer DNA sequence, making accidental matches less common.