Chapter 7

Question 1

In your body osteoblasts cells (bone cells) produce bone tissue but cannot store fat, while adipocyte cells (fat cells) store fat but cannot produce bone tissue? Why?

Answer 1

Cell types are differentiated by the amount of RNA, proteins and their modifications.

Osteoblast and adipocytes in your body share the same DNA, but express different genes.

Approximately 30-60% of genes are expressed in any given cell type, which means that a lot of genes are expressed in widely different cell types.

Alternative splicing contributes as well.

Question 2

What is a housekeeping gene and what can it be use for?

Answer 2

A gene expressed in most cell types and encodes genes necessary for basic functions such a transcription, translation, cytoskeleton and so forth.

Housekeeping genes are generally expressed at approximately the same levels across cell type.

Housekeeping genes can normalize expression of a target gene, for example DKK-1, to the expression of a housekeeping gene.

Question 3

How to TFs bind to DNA?

Answer 3

TFs recognize the major groove surface using multiple interactions (hydrogen, ionic and hydrophobic) rather than the actual base pairs. However, sequence specificity is achieved as the surface features are sequence-dependent. TFs use different structural motifs, commonly involving either α-helices contacting DNA (HTH, Homeodom ain, bZip, HLH), zinc fingers or β-sheets.

Question 4

A motif is recognized by a TF (recepter). The genomes has several motifs, but the receptor never bind more than 10% of the motifs.

What determines which motifs are bound by the receptor?

Answer 4

Transcription factor cooperativity - several factors working together. It can be achieved by dimerization or by other types of protein-protein interactions and it helps to increase specificity as a larger surface is required.
This decreases the randomness.

Another contributor is DNA accessibility. A binding site may be located in nucleosome dense regions.

Cooperativity between TFs can also help overcome these boundaries by chromatin remodeling or nucleosome displacement.

Question 5

What is a transcriptional co-regulator and how is it recruited to DNA?

Answer 5

A transcriptional co-regulator is a protein which is recruited to regulatory elements and assists in regulating transcription (remodels chromatin, modifies histone) , but is does not contact the DNA directly. Co-regulators typically bind indirectly to DNA by tethering to other proteins such as transcription factors.

Question 6

What sort of mechanisms can activators use to increase transcription?

Answer 6

Transcriptional activators …

… promote binding of additional transcriptional regulators.

… recruit and position the RNA polymerase

… release a positioned polymerase, initiating transcription.

…release stalled polymerases.

Question 7

Describe different mechanisms by which transcriptional repressors can decrease transcription?

Answer 7

Signal is turned off
- TFs inactivated.

TFs or co-repressors can recruit chromatin remodelers or histone modifiers to create a more closed chromatin structure, which does not allow transcription.

Another mechanism for gene repression is squelching, in which competition for co-activators between transcription factors results in re-distribution of co-activators from existing enhancers to newly formed enhancers

Question 8

8) How is developmental control of gene expression achieved?

Answer 8

The specific combination of transcription factors and their activities determine developmental control.

For example: In drosophila embryos (single giant cell, multiple nuclei) cis-regulatory elements of the Eve gene is recognized by both transcriptional activators and repressors. The transcriptional outcome is determined by the concentrations of these transcriptional regulators.

Question 9

How does eukaryotes regulate their gene expression?

Answer 9

Chromatin is repressive in nature

Transcriptional regulation depends on activation by making the DNA accessible.

Histone modification and transcription factors with strong motifs can penetrate heterochromatin and facilitate binding of additional TFs.

This facilitates co-factor recruitement and transcriptional activation. Is dependent on several distal regulatory regions(enhancers) and Mediator complex bridging 3d structure between enhancer and promotor.

Question 10

How does prokaryotes regulate their gene expression?

Answer 10

Repressive structure,

Transcriptional regulation of prokaryotes is highly dependent on repressive functions.

In contrast to eukaryotes, they rely on proximal promotor regions. An example would be the LacZ operon.

Question 11

What is an operon?

Answer 11

A functioning unit of DNA containing a cluster of genes under the control of a single promoter.

Question 12

What is a locus?

Answer 12

Fixed position on a chromosome, like the position of a gene or a marker (genetic marker)

Question 13

What is an IPS cell?

Answer 13

iPS cells stand for Induced Pluripotent Cells, and are non-stem cells which have been dedifferentiated by expression of master transcriptional regulators of pluripotency

(the so-called Yamanaka factors)

Question 14

What is cell memory?

Answer 14

Cell memory is the ability of daughter cells to know what genes the parent cells expressed and thereby maintain the same cell type

Question 15

What mechanisms help maintain cell memory?

Answer 15

It can be maintained by logical loops, such as positive feedback where TF A activates its own expression. This means that when proteins are distributed between daughter cells, the expression of A will be maintained

Question 16

Why are genes close to sites occupied by various TFs higher expressed than genes near single TF binding events?

Answer 16

The more TFs bind, the more co-regulators are recruited.

Each TF itself is capable of interacting with various co-regulators, therefore more TFs lead to more recruitment in quantity and diversity of the TFs.

The increase in cofactor load will increase in activity.

Question 17

How is eukaryotic DNA methylated?

Answer 17

DNA can be methylated on cytosine in the sequence CpG.

DNA methylation is typically present on both strands due to an enzyme called maintenance methyl transferase, which recognizes a methylated cytosine on one strand and methylates the paired sequence on the other strand.

Question 18

How is inheritance in a daughter cell ensured?

Answer 18

By DNA methylation

This ensures inheritance, such that after replication one strand in each daughter cell will be methylated in the same pattern as the parent cell.

Question 19

Describe how DNA methylation can repress transcription and the role of reader and writer proteins in this

Answer 19

One way by which DNA methylation represses transcription is by directly interfering with DNA:protein interactions.
Methyl groups on methylated cytosine bases lie in the major groove where transcription factors often contact the DNA. By blocking binding of transcription factor needed for initiation of transcription, methylation can repress gene transcription.

Furthermore, methylated CpG recruits Methyl CpG binding proteins (readers) that act as scaffold proteins for co-repressor complexes containing histone modifying enzymes such histone deacetylases and DNA methylases (writers). This leads to spreading of the repressive marks because neighboring nucleosomes and CpGs are modified and this results in repression of gene loci.

Question 20

How is the methylation status correlated to differentiation capacity? How can a nuclei with highly methylated DNA in nuclei transplantation assays give rise to a new organism?

Answer 20

Methylation status and different capacity are inversely correlated. The more specialized a cell is, the more DNA methylation are present. Methyl transferases methylate DNA, while other proteins are capable of demethylating DNA. Thus, under the right circumstances DNA methylation can be reversed to increase a cells differentiation capacity

Question 21

What is genomic imprinting and what mechanisms contribute to genomic imprinting?

Answer 21

Genomic imprinting is the process whereby only either the maternal or the paternal allele is expressed and the other is silenced. DNA methylation helps to control this by a variety of mechanisms. E.g. methylation of a promoter can silence the gene or it can silence an insulator such that enhancers can work across insulators

Question 22

What is X chromosome inactivation and what is the mechanism of inactivation?

Answer 22

One of the X chromosomes in fetal embryos (at 100 cell stage) is randomly selected and silenced, and maintained throughout life as silent.

A lncRNA called Xist is expressed from a locus on the X chrosome. Xist spreads to the majority (90%) of the X chromosome recruits co-repressors and induces a stable repressive chromatin structure

Question 23

What is a riboswitch? In which cells are they common?

Answer 23

Riboswitches are short RNA sequences present in the growing mRNA which form secondary structures.

Riboswitches are most common in bacteria.

Question 24

How do riboswitches help the cell to adjust gene expression to levels of specific metabolites?

Answer 24

Upon binding of a small molecule riboswitches can alter conformation to affect positively or negatively transcription or translation of the mRNA.

They thus represent a way for the cell to adapt gene expression to the presence of specific metabolites.

Question 25

How can two cells express different isoforms of the same gene?

Answer 25

Alternative splejsing – Contributes to protein diversity.

Question 26

8) In response to stimuli, B lymphocytes switch from expressing a membrane bound antibody to expressing a secreted version of the antibody. Describe how B lymphocytes make this switch in expressing antibody variants. Why are levels of CstF (cleavage-stimulating factor) important for the switch?

Answer 26

The gene encoding the antibody has two RNA cleavage sites/polyadenylation sites which are recognized by the cleavage-stimulating factor (CstF) – an upstream weak binding site and a downstream strong binding site.

In unstimulated B lymphocytes where CstF levels are low, only the strong downstream binding site is bound by CstF and a long transcript which encodes the membrane bound variant of the antibody is formed.

Stimulation of B lymphocytes leads to upregulation of CstF, thereby increasing RNA cleavage at the upstream weak site.

This results in increased levels of the short transcript encoding the secreted version of the antibody.

Thus, by controlling CstF levels, the cell can control which RNA cleavage site is used and therefore which antibody variant is expressed.

Question 27

What is RNA editing, which types of RNA editing exist?

Answer 27

RNA editing is an alteration in the nucleotide sequence of a transcript.

There are two types - the common A-to-I and the less common C-to-U editing. A-to-I is very prevalent in humans and is controlled by formation of self-complementary stem loops in transcripts.

This signifies editing by the ADAR enzymes.

Question 28

What is a nucleotide?

Answer 28

Building blocks of nucleic acids.

They are composed of three subunit molecules: a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

Question 29

How can RNA editing give rise to new isoforms?

Answer 29

Editing can change the amino acid sequence of the resulting product or it can lead to the formation of a new stop codon to allow production of a truncated form of the protein

Question 30

What do it mean when a protein in truncated?

Answer 30

Elimination of the N- or C-terminal portion of a protein by proteolysis or manipulation of the structural gene, or premature termination of protein elongation due to the presence of a termination codon in its structural gene as a result of a nonsense mutation.

Question 31

What is mRNA localization, and why is it useful for the cell? What determines where RNA´s are transported?

Answer 31

mRNA localization is the process where an mRNA is transported to a specific compartment of the cell prior to being translated, rather than being translated immediately after nuclear export.

The advantages are e.g. that this allows proteins to be produced where they are needed, that it allows for cytosolic asymmetry to be generated, and it allows for protein expression to be regulated independently in different regions of the cytosol.

Signals determining the localization of a RNA is often present in the 3’UTR (from stop codon to start of poly(A) tail).

Question 32

Desribe leaky scanning

Answer 32

Initiation can happen at different start codons, as the nucleotides surrounding the start site does not allow proper recognition. This can also be regulated by the different elongation factors which are more or less promiscuous

Question 33

Describe uORFs

Answer 33

These are non-function sequences in the 5’ UTR that can trap a ribosome and thereby block translation. Regulation of how efficiently these are recognized allows regulation of translation initiation

Question 34

Describe IRES

Answer 34

These sequences produce different start sites by adapting a conformation (and using different elongation factors) that allow alternative initiation

Question 35

What is the common nominator for the three concepts; Leaky scanning, uORFs and IRES?

Answer 35

Regulation of translation

Question 36

12) What are the two mechanisms that control mRNA degradation in the cytosol?

Answer 36

RNA can be degraded by decapping and subsequent rapid 5’ →3’ exonucleolytic degradation. In addition, the stability of all mRNAs are regulated by poly(A) tail shortning followed by continued 3’-5’ degradation.

Question 37

What determines mRNA stability?

Answer 37

mRNA stability is controlled both by specific sequences in the 3’ UTR, at which specific proteins bind to increase or decrease the rate of poly A-tail shortening, and by the rate of translation, which uses the poly(A) tail and therefore competes with degradation.

Question 38

What are CpG islands? (CpG dense regions)

Answer 38

short stretch of DNA in which the frequency of the CG sequence is higher than other regions.

“p” signifies that C and G is connected through a phosphodiester bond

(Are actively kept in an unmethylated stage)