Regulation of Gene Expression in Eukaryotes

The two main factors that determine transcriptional regulation are

interactions of cis and trans regulators and chromatin state

interactions of cis and trans regulators refer to

the coordination between DNA sequences and regulatory proteins with the physical transcriptional machinery (RNA polymerase II)

chromatin state refers to how

the physical organization of the genome determines if, when, and where genes are accessible to be transcribed

post-transcriptional regulation occurs

after the mRNA transcript has been produced

mechanisms of post-transcriptional regulation includes

alternative splicing, mRNA stability control, and RNA interference

translational regulation deals with factors affecting

the synthesis of proteins

translational regulation specifically affect

differential rates of translation initiation and elongation efficiency

post-translational regulation finalizes

modifications after a polypeptide is formed

post-translational regulation include

protein folding, chemical modifications of amino acids, protein sorting/trafficking, and protein degradation

trans-acting factors are also known as

transcription factors (TFs)

TFs possess specific structural motifs that allow them to

bind directly to the DNA molecule

TFs attach at

transcription factor binding sites (TFBS)

Parts of the TF proteins initiate transcription by interacting

directly or indirectly with RNA Pol II and associated general TFs at the promoter

TFs that promote or increase gene expression (activators) often

stabilize the RNA Pol II complex at the promoter through mediator proteins

TFs that inhibit or reduce gene expression (repressors) operate by

• competitive binding

• interacting with bound activators to prevent their function

• directly interacting with RNA Pol II or initiation TFs to block the process

cis-regulatory sequences are

DNA sequences where TFs bind to control nearby genes

cis-regulatory sequences are unique in the sense that they are

orientatiion-independent: can function even when flipped (5’ → 3’)

cis-regulatory sequences are found

upstream, downstream, or within introns

[they get with everyone]

immediately adjacent to the transcription state site is the

promotor, which contains the TATA box or other sequences required to recruit RNA Pol II.

enhancers are

cis-regulatory sequences that promote transcription when bound by activator TFs

silencers are

cis-regulatory sequences that repress transcription when bound by repressor TFs

multi-functional sequences are

cis-regulatory sequences that act either as enhancers or silencers depending on the combination of activators and repressors currently bound.

in order to identify cis-regulatory DNA/sequences in different species, researchers use 2 main computational methods:

1. identifying evolutionary conservation in non-coding regions across species

2. predicting clusters of binding sites for specific transcription factors— scan genome for unusual dense binding motifs

in order to identify cis-regulatory DNA/sequences in different species, researchers use 2 main experimental methods:

mutational analysis and reporter gene constructs

in mutational analysis, researchers

mutate a specific non-coding region and measure the resulting gene expression. if mutation reduces expression, the region likely contains an enhancer. if it does the opposite, it likely contains a silencer.

in reporter gene constructs, researchers create

a fusion gene with a functional promoter and a visible reporter (e.g. protein producing color/fluorescence). putative enhancers are placed upstream to see if they drive expression.

a researcher uses reporter gene construct to identify cis-regulatory DNA. Two regions are tested and neither alone drives expression, but together they do. This means

they are both necessary but not individually sufficient

genes can be regulated independently in different tissues by

using different enhancers

The Drosophila Yellow Gene 9which controls the formation of black melanin in the fly’s cuticle, wings, and mouthparts) are controlled by

separate tissue-specific enhancers, in which different sets of TFs present in the tissues bind to respective enhancers to drive expression at specific times and locations

nucleosomes are composed of

eight histone proteins (H2A, H2B, H3, H4) wrapped with about 146 base pairs of DNA

the highest level of DNA compaction is in the form of a

chromosome

chromatin can either be

euchromatin (open chromatin) or heterochromatin (closed chromatin)

euchromatin appears as

light bands in G-banded karyotypes. they are accessible for RNA Pol II, and transcription can occur

heterochromatin appears as

dark bands in G-banded karyotypes. They are highly condensed, so transcription is unable to occur

controlling the positioning and density of nucleosomes to allow access to promotors are done by

chromatin remodeling complexes

functions of chromatin remodeling complexes include

nucleosome sliding, nucleosome eviction, nucleosome assembly/spacing, and histone replacement

in histone replacement,

standard histones are replaces with variants that are easier to displace

SWI/SNF is responsible for

sliding/ejection for opening

SWR1 is responsible for

histone replacement for opening

ISWI is responsible for

positioning for closing

in histone tail modifications,

post-translational chemical groups are added to amino acids on histone tails

the histone code hypothesis refers to how

combinations of modifications (methylation & acetylation) have specific meanings for chromatin stability

DNA methylation primarily occurs in

CpG dinucleotides

clusters of CpG sites (aka CpG islands) are usually found at

promoters

unmethylated CpG islands are

open chromatin and transcription occurs

methylated CpG islands are

closed chromatin and transcription is repressed

genomic imprinting is a form of

epigenetic regulation in which gene expression depends on the parent of origin— offspring will selectively methylate/silence either the maternal or paternal copy of a gene

imprinting reset refers to how

germ-line imprints are erased during development and re-established during gametogenesis

in post-transcription regulation there are 5 types of alternative splicing:

1. exon skipping/inclusion

2. alternative 3’ splice sites

3. atternative 5’ splice sites

4. mutually exclusive exons

5. intron retention

through alternative splicing rat a-tropomyosin gene produces

9 distinct mature mRNAs across muscle, brain, and fibroblast cells

in drosophila, sex is determined by the proteins

transformer and transformer 2

in female drosophilia, the proteins, transformer and transformer 2 are

produced. These proteins bind to exon 4, recruiting splicing factors to its 3’ splice site, resulting in a female-specific RNA

4 mRNA stability factors include

1. poly(A) tail length

2. sequences in the 3’ untranslated region (UTR)

3. chemical/hormonal factors

4. small regulatory RNAs (siRNAs and miRNAs)

RNA interference (RNAi) refers to how

dsRNA destroys endogenous (growing) mRNA

in RNAi, the dsRNA is first

cleaved into fragments. RISC (RNA-Induced Silencing Complex) then binds the fragments and unwinds it to keep a single-stranded guide strand. It then targets complementary mRNA, in which they are either cleaved/degraded, translated or repressed, or sequestered in P-bodies

miRNAs usually have

imperfect base-pairing and results in translation block or degradation

siRNAs usually have

perfect base-pairing and results in mRNA degradation

this regulatory RNA is encoded in the genome, is transcribed by RNA Pol II, and produce hairpins

miRNA

this regulatory RNA can be exogenous or endogenous (growing) and start as long dsRNA

siRNAs

dosage compensation functions to

equalize gene expression between sexes in organisms with chromosomal sex determination

mammalian X-inactivation initiates at the

X inactivation center (XIC) and spreads

the inactive X is coated by the XIST (X inactive specific transcript) gene produces

a functional non-coding RNA produced by the XIST (X inactive specific transcript) gene

in dosage compensation, the chromosome

condenses into a Barr body

drosophilia hypertranscription occurs in

XY males

the single X chromosome is bound by

MSL (male-specific lethal) protein complex and roX RNAs

the MSL (male-specific lethal) protein complex modifies

histones to open the chromatin, increasing transcription from the male X chromosome by two-fold