1
2
3
4
Once obsolete proteins are tagged with at least four ubiquitin molecules, they
are destroyed by proteasomes. Proteasomes are voracious protein shredders,
but the destructive machinery is carefully protected so that it can't attack all of
the normal proteins in the cell. The proteasome, shown here from PDB entry
1fnt, is shaped like a cylinder, with its active sites sheltered inside the tube. The
caps on the ends regulate entry into the destructive chamber, where the protein
is chopped into pieces 3 to 23 amino acids long.
Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is
performed by a nonspecific and abundant barrel-shaped complex called the 20S
proteasome. Substrates access the active sites, which are sequestered in an
internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked
in the unliganded 20S proteasome by amino-terminal sequences of alphasubunits.
Peptide products probably exit the 20S proteasome through the same
opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are
heptamers that stimulate 20S proteasome peptidase activity in vitro and may
facilitate product release in vivo. Here we report the co-crystal structure of yeast
20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26
carboxy-terminal tails provide binding affinity by inserting into pockets on the
20S proteasome, and PA26 activation loops induce conformational changes in
alpha-subunits that open the gate separating the proteasome interior from the
intracellular environment. The reduction in processivity expected for an open
conformation of the exit gate may explain the role of 11S regulators in the
production of ligands for major histocompatibility complex class I molecules.
(PDB, Whitby et al., (2000) Nature 408: 115-120,
http://www.rcsb.org/pdb/explore/explore.do?structureId=1fnt).
5
Regulation of the chromatin structure represents one of the very basal gene
expression regulatory levels. Chromatin is a substrate for DNA-dependent RNA
polymerases that transcript the DNA encoded information into the “words and
sentences” of RNA.
Regulation of chromatin structure and its accessibility to DNA-dependent RNA
polymerases depends on many factors, one of the most important is the
regulation of chromatin binding to nucleosomes and chromatin methylation.
Regulation of chromatin interaction with histones, the positively charged
proteins forming the core of nucleosomes, is performed via modification of
acetylation status of the N-terminal portion of histones, especially histones H3
and H4. This occurs via action of histone acetyl transferases or histone
deacteylases.
6
Modification of the chromatin methylation is performed via DNA methyltransferases.
Interestingly, there is difference in the methylation in animals and in plants.
In animals, the methylation takes place mostly on the cytosine that occurs next to
guanosine (the sequence is denoted as CpG). In mammals, 60-90% of all CpGs are
methylated.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and
asymmetrically (CpNpNp), where N is any nucleotide.
Methylation status is usually “reset” in the zygote and is reconstituted during
development again. E.g. the methylation is very low in the mouse embryo at the blastula
stage, however, DNA derived from later stages when organogenesis is initiated is
substantially more modified by methylation.
DNA methylation also stably alters the gene expression pattern in cells such that cells
can "remember where they have been"; in other words, cells programmed to be
pancreatic islets during embryonic development remain pancreatic islets throughout the
life of the organism without continuing signals telling them that they need to remain
islets.
DNA methylation is involved in the genomic imprinting, i.e. the genes originating from
both parents are often diversely methylated, which results into differential expression of
parental genomes (for the importance of the imprinting in the parental conflict and
epigenetics, see the lecture “Bi0580 Developmental genetics” by prof. Vyskot).
Up to know it is not clear how methylation regulates transcription. Possibly, methylation
status affects chromatin configuration or binding general repressor factors.
7
8
Regulation of transcription occurs via specific interaction of both general and
tissue specific transcription factors (TFs) with promoter and/or enhancer
sequences.
The scheme above shows simplified subsequent formation of the complex of TFs
involved in the regulation of transcription. Interaction of general TFIID with the
TATA box induces distortion of the DNA structure (see the next slide).
9
Induction of structural changes upon interaction of TFIID with DNA. This may be
important for the assembly of other TFs involved in the formation of
transcription initiation complex.
This change of confirmation provides a kind of “signature” that is recognized by
other proteins and NA polymerase to recognize the proper binding site. However,
there are also TATA box-less promoter, where probably other types of
“signatures” occur.
10
The scheme showing the formation of the transcription initiation complex and
the interaction of both positive (open symbols) and negative (solid symbols)
factors.
These proteins bind to the regulatory sequences that might be hundreds or even
thousands of base pairs away from the promoter. These protein interact with
each other and with the RNA polymerase, integrating thus many signals into a
“yes” or “no” response of the basal promoter, i.e. the region adjacent to the TATA
box and recognized by the RNA polymerase.
The individual positive or negative factors are complex and their activity might be
regulated by their phosphorylation status or via their interaction with other
proteins (i.e. momomeric or dimeric) etc..
11
There is a whole family of transcription activating factors (TAFs) that interact with
signalling molecules, e.g. steroid hormones, thyroid hormones or retinoic acid
and in a response to the signal transfer to the nucleus where they regulate
transcription.
One of the type of TAF are leucine zipper or bZIP type TAFs. These TAFs are
dimeric, with leucine-rich hydrophobic face formed by the Leu that occurs every
7th aa.
That allows the factor to take the proper configuration, which provides the dimer
with the ability to bind DNA via charged aa.
12
An example of the “microprocessor”-like acting promoter is a promoter of the endo16
gene from the sea urchin.
There have been identified several gene regulatory modules in the endo16 gene that
have positive or negative regulatory role. These modules were identified via formation
of deletion mutants of the transcriptional fusions with reporter gene.
The analysis has revealed that the module A has a positive function and must interact
with its cognate TAFs for transcription to occur.
Module G enhances the expression when the A and B are active.
C, D, E and F are responsible for the specificity of the expression of endo16 during sea
urchin development.
Each of the modules has several protein interaction sites, some of them general, other
unique. Site for the protein SpGCF1 is present in many modules and is probably
responsible for looping of chromatin, allowing thus bringing of distal regulatory modules
close to the basal promoter.
This type of regulation, i.e. based on the different activities of diverse regulatory
sequences is sometimes called combinatorial and is common for development of many
living creatures.
In the combinatorial type of regulations, some modules may act synergistically, some of
them antagonistically, some may have both positive and negative roles (e.g. the module
B, see the figure). This variability allows very precise and responsive regulations towards
changing environmental conditions.
13
An example of the combinatorial gene regulation is the regulation of β-globin type of hemoglobin chains
of humans.
As discussed in the Lesson 5 (course Bi8940 Developmental biology), the type of hemoglobin produced by
the fetus changes during development. The hemoglobin present in the liver-produced hemoglobin is
composed of two α- and two β-type chains. The β-type hemoglobin chains are of several developmental
types, produced by ε, γ1, γ2 and β (in this order). In addition, there is minor adult type of β-type
hemoglobin, called δ globin.
The genes for the β-type chains are aligned on the chromosome in the order, in which they are expressed
during development (see the figure).
For the expression of individual cell types is distinctive an upstream regulatory sequence called locus
control region (LCR). LCR is located about 50 kbp away from the most proximal ε gene.
The LCR structure is different in erytrocyte precursor cells in comparison to other cells that could be
demonstrated by the changes in the sensitivity to low concentrations of DNase, suggesting low amount of
nucleosomes bound.
For the expression of the particular genes, the interaction of their regulatory sequences with LCRs is
necessary. Because of LCR can interact only with one regulatory sequence at a time, only one type of
genes for the particular β-type chain is activated (the first interaction of LCR with ε gene, which is later in
development replaced by the other one, is shown by the double-headed arrow).
The underlying molecular mechanisms of the specific pattern of the LCR movement from the most
proximal towards the most distal gene cannot be satisfactory explained.
Probably, acetylation of H3 histones might play a role and possibly, other genes outside of the β-type
chain family are involved in the regulation of LCR activity. That seems to be confirmed by the
identification of other human genes with similar structure, suggesting common regulatory mechanisms
via LCRs.
For
14
15
16
Myelin basic protein (MBP) is a protein believed to be important in the process of
myelination of nerves in the nervous system.
The images show localization of mRNA for MBP. Digoxigenin-labeled MBP RNA
was microinjected into mouse oligodendrocytes growing in primary culture. The
injected RNA appeared as small granules which were present throughout the
cytoplasm and processes, and was also found dispersed in the peripheral
membranes of the cell.
To analyze the three dimensional distribution of microinjected labeled MBP
mRNA throughout the cell, consecutive optical sections through a single
oligodendrocyte were collected, reconstructed, and visualized using volume
rendering (Fig. A) or isosurface rendering (Fig. B) techniques. An oligodendrocyte
microinjected with MBP mRNA, visualized by volume rendering is shown in Fig. A.
RNA granules were observed throughout the perikaryon and in some, but not all,
processes. The granules in the perikaryon and in the processes appeared to be
equivalent in size. In some regions the granules in the processes were aligned in
tracks. Although not apparent from this image, the nucleus was devoid of
granules.
17
Another well studied example of the diffusion-entrapment mechanism is the
Xenopus Xcat-2 mRNA, which encodes a Nos related zinc-finger RNA-binding
protein.
During the early stages of Xenopus oogenesis, Xcat-2 mRNA is restricted to a
specific structure in the cytoplasm called the mitochondrial cloud (MC). The
mitochondrial cloud, also called Balbiani body, consists mostly of mitochondria
and small vesicles, and is the source of germinal granule material [32]. The
movement of the MC in the cytoplasm results in the localization of the Xcat-2
mRNA at the vegetal cortex (Shahbabian and Chartrand, 2012).
18
Localized stabilization of a transcript is another mechanism by which an mRNA can be
subcellularly targeted. In this case, an mRNA is rapidly degraded in most parts of the cell, but it is
protected from degradation at a specific location. The hsp83 mRNA, which encodes a heat shock
protein in Drosophila, is a well-characterized example of this kind of localization (Fig. 2b). This
transcript is localized at the posterior pole of the early Drosophila embryo by the selective
stabilization of the mRNA at the posterior pole and degradation of the transcript elsewhere in
the cytoplasm.
The level of hsp83 mRNA, which is a maternally encoded transcript, decreases more rapidly in
embryos than in unfertilized eggs, which suggests that two separate mechanisms control the
stability of this transcript [38]. These two independent pathways, which are called ‘‘maternal’’
and ‘‘zygotic’’ pathways, use maternally and embryonic encoded proteins, respectively, to
degrade the hsp83 transcript [38]. By analyzing the 3’UTR of hsp83 mRNA, a region from
nucleotides 253–349 was identified as the Hsp83 degradation element (HDE), which directs the
destabilization of this mRNA in unfertilized eggs. However, this region has no effect in the
zygotic degradation pathway, and transcripts without the HDE domain are subject to
degradation by the embryonic degradation machinery [38]. The hsp83 ORF has also been shown
to affect the stability of the transcript. A region at the 3’ end of the ORF, which comprises 615
nucleotides, has been found to be responsible for this destabilization, and was consequently
called Hsp38 instability element (HIE) [39]. This region, which has the major effect in the
destabilization of the transcript, functions together with the HDE for complete degradation. The
HIE domain contains six stem-loop structures that are recognized by the maternally encoded
RNA-binding protein Smaug [39, 40]. It was shown that in Smaug mutants, degradation and thus
localization of hsp83 mRNA are impaired. Smaug recruits the CCR4/POP2/NOT deadenylase
complex, triggering deadenylation and thus degradation of the hsp83 transcript [40]. Although
Smaug is present throughout the pole plasm, the hsp83 mRNA is protected from Smaug action at
the posterior pole. This protection is related to a 57 nt region in the 3’UTR (nucleotides 351–
407) downstream of HDE, which is called HPE (Hsp83 protection element). HPE is sufficient to
confer stability to an unstable transcript at the pole plasm [40]. The mechanism by which this
domain functions is not clear, and may include interaction of trans-acting factors that block the
availability of the transcript to Smaug (Shahbabian and Chartrand, 2012).
19
Localization of ASH1 mRNA is essential for the asymmetric distribution of Ash1, which acts as a transcriptional
repressor of the HO endonuclease and results in inhibition of mating-type switching in daughter cells [88, 89]. The
ASH1 mRNA contains four localization elements, three in the coding sequence (E1, E2A, and E2B) and one overlapping
the end of the coding sequence and the 3’UTR (E3) [25, 90]. While the presence of these four elements leads to an
optimal localization, deletion analysis revealed that each element is sufficient for localization of a reporter mRNA to
the bud.
When each of these elements was inserted in multiple copies in the 30UTR, the new constructs showed nearly normal
localization. However, for these mRNAs, the asymmetric distribution of Ash1 was impaired, suggesting that the
position of these elements is important for Ash1 sorting but not for ASH1 mRNA localization [91]. Although the
primary sequences of the four ASH1 localization elements are different, they all fold into a stem-loop structure that
contains a few conserved nucleotides [92, 93]. All four elements interact with the same RNA binding protein called
She2, which is involved in the localization of bud-localized mRNAs in S. cerevisiae.
She2 forms a tetramer under physiological conditions, and mutations that disrupt this tetrameric state abolish its
RNA-binding capacity and impair She2-dependent localization to the bud tip [94]. She2 interacts directly with the Cterminal
domain of She3, an adaptor protein that links the She2–mRNA complex to the molecular motor Myo4 (Fig.
2c) [55, 95, 96]. Recent evidence also suggests that She3, besides its role in connecting the She2–RNA complex to
Myo4, is itself able to bind RNA and acts synergistically with She2 to increase the affinity and specificity of RNA
binding [97].
Recent studies on Myo4 helped to explain why multiple localization elements are required for proper ASH1 mRNA
localization. Myo4 is a class V myosin whose main function is the transport of mRNAs to the bud tip using actin
filaments [98–100]. Myo4, unlike other type V myosins, is a nonprocessive monomer in vivo, but it becomes
processive when present in the form of oligomers [101, 102]. Purification of the localization complex associated with
a single localization element revealed that multiple copies of Myo4 are associated with this RNA [103]. Moreover,
increasing the number of Myo4 attached to the ASH1 mRNA increased the efficiency of localization of this transcript.
These results suggest that each localization element interacts with higher order protein complexes in which a She2
tetramer may recruit multiple copies of Myo4, thus ensuring a continuous and processive movement of the mRNP
complex into the bud. Moreover, it is possible that a She2 tetramer binds simultaneously to the localization elements
of a single transcript or, alternatively, to those of different mRNAs. This would bring multiple mRNAs together within
a single complex in which several Myo4 molecules modulate their transport to the bud tip (Shahbabian and Chartrand,
2012).
20
21
22
Scheme of the auxin signaling pathway as an example of the role of protein
stabilization leading to regulation of gene expression.
Under low intracellulr auxin concentrations, the transcription activators of auxinregulated
genes, which are called auxin responsive factors (ARFs), are in a
complex with negative regulators of transcription, so called AUX/|IAA proteins. In
the complex, Arfs can not activate transcription.
After auxin is imported into the cell, it binds to the TIR1 protein, that allows
interaction with AUX/IAA-ARF complex and targets AUX/IAA protein for the
degradation via proteosome.
That allows ARFs to enter nucleus and activate trancription of auxin-induced
genes.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48