Transcript the blue ball

Molecular Biology
Medicine Part II b
SWITH
Ettore Sansavini Health Science Foundation – ONLUS
Lugo (Ravenna), Italy
Carlo Ventura
Professor of Molecular Biology
University of Bologna, Italy
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Gene Expression
The analysis of specific patterns of gene expression is major
challenge in the understanding of complex processes including
cell proliferation and differentiation, as well as tissue organization
into a specific architecture. It is now increasingly becoming
evident that genomic sequence represents only one level of
genetic complexity and that disclosing the ordered and timely
patterning of gene expression involves another level of
complexity of equal magnitude in the definition and biology of
living organisms
Eukaryotic RNA polymerases
Type
Location
Cellular transcripts
Effects of 
amanitin
------------------------------------------------------------------------------------------------------I
Nucleolus
18S, 5.8S, and 28S rRNA
Insensitive
II
Nucleoplasm
mRNA precursors and snRNA
Strongly inhibited
III
Nucleoplasm
tRNA and 5S rRNA
Inhibited by high
concentrations
-------------------------------------------------------------------------------------------------------
Factor
(Abbreviation) Composition
TFIIA (IIA)
TFIIB (IIB)
TFIID (IID)
2 or 3 subunits
single subunit
TBP (TATA boxbinding protein)
Function
stabilizes binding between TFIID and promoters
interaction between TFIID and polII-TFIIF
binding to TATA box
8 - 10 TAFII's (TBP-associated proteins) interaction with promoter elements and with
gene-specific transcription factors
TFIIF (IIF)
2 subunits
somewhat like sigma in prokaryotes, this protein
causes RNA pol II to bind to the complex
assembly at the promoter
TFIIE (IIE)
2 subunits
required for binding and stimulation of
transcription
complex
kinase activity (associated kinase activates polII
by phosphorylation), helicase activity
TFIIH (IIH)
A complex of CPSF (cleavage and
polyadenylation specificity factor) (the
blue ball), CF (cleavage factors) I and
II (the brown balls) and CstF
(cleavage stimulation factor) (the gray
ball) bind to these sequences.
A cleavage occurs and CPSF
remains and is joined by PAP (poly
[A] polymerase) (the red ball).
PAP begins the synthesis of poly
[A], resulting in the addition of the
first 10 A's.
Finally, PAB II (pol [A] binding protein
II) joins the reaction, stimulating
the synthesis of poly [A], extending
the tail t about 200 A residues.
RNA editing of apo-B pre-mRNA. The apo-B mRNA produced in the liver has the
same sequence as the exons in the primary transcript. This mRNA is translated into
Apo-B100, which has two functional domains: a N-terminal domain (green) that
associates with lipids and a C-terminal domain (orange) that binds to LDL receptors on
cell membranes. In the apo-B mRNA produced in the intestine, the CAA codon in
exon 26 is edited to a UAA stop codon. As a result, intestinal cells produce Apo-B48,
which corresponds to the N-terminal domain of Apo-B100. [Adapted from P. Hodges
and J. Scott, 1992, Trends Biochem. Sci. 17:77.]
RNA editing is not confined to apolipoprotein B. Glutamate opens cation-specific
channels in the vertebrate central nervous system by binding to receptors in
postsynaptic membranes. RNA editing changes a single glutamine codon (CAG) in
the mRNA for the glutamate receptor to the codon for arginine (read as CGG). The
substitution of Arg for Gln in the receptor prevents Ca2+, but not Na+, from flowing
through the channel. RNA editing is likely much more common than was previously
thought. The chemical reactivity of nucleotide bases, including the susceptibility to
deamination has been harnessed as an engine for generating molecular diversity at
the RNA and, hence, protein levels.
REGULATION OF EUKARYOTIC GENE EXPRESSION
TRANSCRIPTION FACTORS
General Transcriptional Machinery
Zinc Fingers
Zinc Fingers and DNA Bending
hsp90
R
R
R
R
R
R
hsp90
NHR and DNA Bending
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Reflected light-sheet microscopy (RLSM)
46
(B) Maternal effect genes. The anterior axis is specified by
the gradient of Bicoid protein (yellow through red). (C) Gap
gene protein expression and overlap. The domain of
Hunchback protein (orange) and the domain of Krüppel
protein (green) overlap to form a region containing both
transcription factors (yellow). (D) Products of the fushi
tarazu pair-rule gene form seven bands across the embryo.
(E) Products of the segment polarity gene engrailed, seen
here at the extended germ band stage.
Histones H2A and H2B are yellow and red, respectively; H3 is purple, and H4 is
green. The DNA helix is wound about the protein core. The histone tails extend from
the core and are the sites for acetylation, which may disrupt the formation of
nucleosome assemblages.
(a) Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) of the repressor Ume6 interacts
with a specific upstream control element (URS1) of the genes it regulates. The Ume6 repression domain (RD) binds Sin3, a subunit
of a multiprotein complex that includes Rpd3, a histone deacetylase. Deacetylation of histone N-terminal tails on nucleosomes in the
region of the Ume6-binding site inhibits binding of general transcription factors at the TATA box, thereby repressing gene expression.
(b) Activator-directed hyperacetylation of histone N-terminal tails. The DNA-binding domain of Gcn4 interacts with specific
upstream-activating sequences (UAS) of the genes it regulates. The Gcn4 activation domain (AD) then interacts with a multiprotein
histone acetylase complex that includes the Gcn5 catalytic subunit. Subsequent hyperacetylation of histone N-terminal tails on
nucleosomes in the vicinity of the Gcn4-binding site facilitates access of the general transcription factors required for initiation.
Repression and activation of some genes in higher eukaryotes occurs by similar mechanisms.
Structure of Histone Acetyltransferase. The
amino-terminal tail of histone H3 extends into a
pocket in which a lysine side chain can accept an
acetyl group from acetyl CoA bound in an
adjacent site.
Structure of a Bromodomain. This four-helix-bundle domain binds peptides
containing acetyllysine. An acetylated peptide of histone H4 is bound in the
structure shown.