Transcript Translation

E. CELL SPECIALIZATION: RNA and
Protein Regulation
1. nRNA to protein (review)
2. Cell-Specific Regulation of mRNA Production
3. Cell-Specific Regulation of Peptide and
Protein Production
1. nRNA to protein (review)
nucleus
cytosol
Fig. 17-5
The Genetic Code
•
20 amino acids
•
64 codons:
end of codon)
end of codon)
First mRNA base (5
Third mRNA base (3
Second mRNA base
•
61 = code for
amino acids
•
3 = stop signals
•
Genetic code is
redundant
(degenerate base)
•
No codon specifies
>1 unique amino
acid
•
Genetic code is
nearly universal (a
few exceptions)
•
Must be read in
frame (like words in
a book)
Fig. 17-13
Key Players in:
Amino
acids
Polypeptide
tRNA with
amino acid
attached
Ribosome
tRNA
Anticodon
Codons
5
mRNA
3
Translation
- mRNA
- tRNA
- ribosome
- amino acids
• Translation determines the primary structure
• Primary structure determines the repetitive folding of the
secondary structure
• Tertiary structure arises due to complex folding
• Quaternary structure arises due to the joining of multiple
peptide chains subunits
• The latter two are the result of post-translational changes
to the primary sequence
Fig. 5-21a
Primary Structure
1
Primary
structure,
the sequence
of amino
acids in a
protein, is like
the order of
letters in a
long word
+H
5
3N
Amino end
Primary
structure is
determined
by inherited
genetic
information
10
Amino acid
subunits
15
20
25
Fig. 5-21c
The coils and folds ofSecondar
secondary
structure
Structure
result from hydrogen bonds between
repeating constituents of the polypeptide
backbone
 pleated sheet
 helix
Fig. 5-21f
Tertiary structure is determined by
interactions between R groups, rather than
interactions between backbone constituents
Hydrophobic interactions and
van der Waals interactions
Hydrogen
bond
Disulfide bridge
Polypeptide
backbone
Ionic bond
Strong covalent
bonds called
disulfide bridges
may reinforce the
protein’s structure
Fig. 5-21g
3 polypeptides
 Chains
Quaternary structure
results when two or
more polypeptide
chains form one
macromolecule
 Chains
Collagen
Hemoglobin
- It is hard to predict a protein’s structure from its primary structure
- Most proteins go through several states on the way to stable structure
2. Cell-Specific Regulation of mRNA Production
a. Co/post-transcriptional RNA modification can
effect amount and type of protein expressed
1. 5’ Capping and 3’ Polyadenylation
determine how the nRNA will be handled
2. Splicing different mRNAs from the same
nRNA using different exons allows cells to
choose the protein they will make
Formation of the 5’ Cap in mRNA
Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008)
The roles of the 5’ Cap
Allows the cell to distinguish mRNA from other RNA
Allows for processing and export of the mRNA
Allows for translation of the mRNA in the cytosol
Formation of the 3’ PolyA tail in mRNA
The position
of the tail is
coded in DNA
Figure 6-37 Molecular Biology of the Cell (© Garland Science 2008)
RNA Pol II reads the DNA and attaches:
- cleavage stimulation factor
- cleavage and polyadenylation specificity factor
RNA is cleaved and Poly-A polymerase added
- ~200 adenosine nucleotides are added
- CstF falls off
Poly-A Binding Proteins are added
- CPSF and Poly-A Pol fall off
- Poly-A binding proteins modify length of tail by
terminating or prolonging Poly-A Pol activity
Figure 6-38 Molecular Biology of the Cell (© Garland Science 2008)
Many proteins have alternative poly-A sites which can
either change the regulation of expression at the 3’UTR
or, less commonly, change the length of the coding region.
The choice of poly-A site
can be regulated by
external signals
The roles of the 3’ Poly-A Tail
Regulates termination of transcription
Regulates nuclear transport
Regulates the initiation of translation
Controls the total amount of translation
2. Splicing different mRNAs from the same nRNA
using different exons allows cells to choose the
protein they will make
– Alternative splicing occurs in ~92% of human genes
– “Splice sites” are formed from consensus sequences
found at the 5’ and 3’ ends of introns
– Different splicosome proteins made in different cells
recognize different consensus sequences
– The result is families of related proteins from the
same gene in different cell types
Fig. 17-10
•RNA splicing removes introns and joins exons,
creating an mRNA molecule with a continuous
coding sequence
5’ Exon Intron
Exon
Exon
Intron
3’
Pre-mRNA 5’ Cap
Poly-A tail
1
30
31
Coding
segment
mRNA 5’ Cap
1
5’ UTR
104
105
146
Introns cut out and
exons spliced together
Poly-A tail
146
3’ UTR
Examples of alternative RNA splicing (Part 1)
Examples of alternative RNA splicing (Part 2)
Alternative RNA splicing to form a family of rat αtropomyosin proteins
The Dscam gene of Drosophila can produce 38,016
different types of proteins by alternative splicing
The proteome in most eukaryotes dwarfs the genome in complexity!
Dscam protein is required to keep dendrites from the
same neuron from adhering to each other
Dscam complexity
is essential to the
establishment of the
neural net by excluding
self-synapses from
forming
Differential RNA Processing
Splicing Enhancers and Recognition Factors
- These work much like transcription enhancers and factors
- Enhancers are RNA sequences that bind factors to promote or silence
spliceosome activity at splice site
- Many of these sequences are cell type-specific, eg. muscle cells have
specific sequences around all of their splice sites, thus make musclespecific variants
- Trans-acting proteins recognize these sequences and recruit or block
spliceosome formation at the site
Muscle hypertrophy through mis-spliced myostatin
mRNA
Splice site
mutations
can be very
deleterious,
rarely can be
advantageous
Fig. 17-11-1
RNA transcript (pre-mRNA)
5
Exon 1
Protein
snRNA
Spliceosomes
consist of a
variety of
proteins and
several small
nuclear
ribonucleoprote
ins (snRNPs)
that recognize
the splice sites
Intron
Exon 2
Other
proteins
snRNPs
Fig. 17-11-2
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Other
proteins
snRNPs
Spliceosome
5
Exon 2
Fig. 17-11-3
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
Differential RNA Processing
Spliceosome proteins link directly to the nuclear
pore to facilitate transfer of the spliced mRNA
into the cytosol
Alternative splicing can have very
powerful effects on protein function
• Proteins often have a modular architecture
consisting of discrete regions called domains
• In many cases, different exons code for the
different domains in a protein
• Exon shuffling may result in the evolution of
new proteins
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-12
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
b. Selective Degradation of RNA
1. Prevention of export of incomplete or intronic
RNA from the nucleus
2. Prevention of translation of damaged or
unwanted RNA in the cytosol
2. Cytosolic selection
Cell type 1
Cell type 2
1. Prevention of export of incomplete or
intronic RNA from the nucleus
– More genes are transcribed in the nucleus than than
are allowed to be mRNA in the cytosol
– The unused nRNAs are degraded in the nucleus or
used to make non-coding RNA molecules
At every step in the processing of the transcript it must lose
and/or gain the appropriate proteins to be identified as ‘ready’.
‘export ready’
Figure 6-40 Molecular Biology of the Cell (© Garland Science 2008)
‘translation ready’
Key identifying proteins:
Positive for export
cap and PolyA binding proteins
exon junction and SR proteins
nuclear export receptor
Negative for export
snRNP
Positive for translation
translation initiation factors
Negative for translation
cap binding protein
The inappropriate combination of markers leads to
degradation by nuclear exosome and cytosolic exonuclease
2. Prevention of translation of damaged or
unwanted RNA in the cytosol
a. Failed recognition of 5’-cap and poly-A tail
prevents translation-initiation machinery
b. Eukaryotes have nonsense-mediated mRNA
decay system to eliminate defective mRNAs,
mainly due to nonsense codon
c. Bacteria also have quality control mechanisms to
deal with incompletely synthesized and broken
mRNAs
Eukaryotic block to translation
Figure 6-80 Molecular Biology of the Cell (© Garland Science 2008)
Prokaryotic block to translation
Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008)
3. Cell-Specific Regulation of Peptide and Protein
Production
a. Regulation of translation
b. Co-/Post-translational protein regulation
a. Regulation of translation
1. 5’ and 3’ untranslated regions of mRNAs
control their translation
2. Global regulation of translations by initiation
factor phosphorylation
3. Small noncoding RNA transcripts regulate
many animal and plant genes
4. RNA interference is a cell defense mechanism
1. 5’ and 3’ untranslated regions of mRNAs
control their translation
a. The primary site of translation initiation is the
5’-cap
b. Internal ribosome entry sites provide
alternative sites of translation initiation
c. Changes in mRNA stability can regulate the
amount of protein translated from mRNA
1. Cytoplasmic poly-A addition can regulate
translation
2. External factors can extend RNA life
a. The primary
site of translation
initiation is the
5’-cap
Figure 6-72 (part 1 of 5) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-72 (part 2 of 5) Molecular Biology of the Cell (© Garland Science 2008)
b. Internal ribosome entry sites provide
alternative sites of translation initiation
• Multiple AUG start codons in one mRNA
sequence
• A given cell can choose one or the other by it
the translation initiation factors it expresses
Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
Fig. 17-10
c. 5’ caps and 3’ poly-A tails dictate the duration
of time that the mRNA is active in the cytosol
5’ Exon Intron
Exon
Exon
Intron
3’
Pre-mRNA 5’ Cap
Poly-A tail
1
30
31
104
105
146
Coding
segment
mRNA 5’ Cap
1
5’ UTR
Poly-A tail
146
3’ UTR
c. 5’ caps and 3’ poly-A tails dictate the duration
of time that the mRNA is active in the cytosol
Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008)
The length of the poly-A tail determines how long the mRNA survives
Once the tail is degraded: Coding sequence is destroyed
and/or
The 5’ cap is removed
Figure 7-110 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-109 Molecular Biology of the Cell (© Garland Science 2008)
2. External factors can extend RNA life
The length of translation can also
respond to external regulation from
hormones, growth factors, etc.
Degradation of casein mRNA in the presence and
absence of prolactin
b. Co-/Post-translational protein regulation
1. Folding and membrane insertion
2. Covalent modifications
3. Polymer assembly
4. Proteolytic modifications
1. Folding and membrane insertion
• Molecular chaperones help guide the
folding of most polypeptides while still
being synthesized
– Heat shock proteins (Hsp)
• Hsp70 (BIP)
• Hsp60 (chaperonins)
– Calnexin, calreticulin
– “Folding”, “Protease Inhibitor”
Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)
Fig. 5-24
Polypeptide
Correctly
folded
protein
Cap
Hollow
cylinder
Chaperonin
(fully assembled)
Steps of Chaperonin 2
Action:
1 An unfolded polypeptide enters the
cylinder from one end.
The cap attaches, causing the 3 The cap comes
cylinder to change shape in
off, and the properly
such a way that it creates a
folded protein is
hydrophilic environment for
released.
the folding of the polypeptide.
Many membrane proteins are associated
with the lipid bilayer during translation
Figure 12-43c Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-47 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Figure Q12-5 Molecular Biology of the Cell (© Garland Science 2008)
Misfolded proteins are controlled by regulated destruction
proteasome
Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-54 Molecular Biology of the Cell (© Garland Science 2008)
2. Covalent Modifications
• Glycosylation by cell-specific enzymes can
change the function of a shared protein
• Different kinases in different cells may
phosphorylate proteins at alternative sites
• Isomerization of disulfide linkages in different
cells can produce different functions
• Variability in methylase/acetylase proteins can
dramatically alter cell phenotype and function
Figure 19-60b Molecular Biology of the Cell (© Garland Science 2008)
3. Polymer Assembly
Figure 3-27a Molecular Biology of the Cell (© Garland Science 2008)
42 genes in humans for -collagen
You need three to make a protein
40 different proteins have been shown
Figure 19-62 Molecular Biology of the Cell (© Garland Science 2008)
4. Proteolytic Modifications
Figure 3-35 Molecular Biology of the Cell (© Garland Science 2008)