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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
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1. nRNA to protein (review)
nucleus
cytosol
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Fig. 17-5
Second mRNA base
Firs
t mR
NA
base
(5�
end
of c
odon
)
Third
mR
NA
base
(3�
end
of c
odon
)
• 20 amino acids
• 64 codons:
• 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)
The Genetic Code
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Fig. 17-13
Polypeptide
Ribosome
Aminoacids
tRNA withamino acidattached
tRNA
Anticodon
Codons 3�5�
mRNA
Key Players in:
Translation- mRNA- tRNA- ribosome- amino acids
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• 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
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Fig. 5-21a
Amino acidsubunits
+H3NAmino end
25
20
15
10
5
1Primary Structure
Primary structure, the sequence of amino acids in a protein, is like the order of letters in a long word
Primary structure is determined by inherited genetic information
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Fig. 5-21c
Secondar Structure
β pleated sheet
α helix
The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone
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Fig. 5-21f
Polypeptidebackbone
Hydrophobic interactions andvan der Waals interactions
Disulfide bridge
Ionic bond
Hydrogenbond
Tertiary structure is determined by interactions between R groups, rather than interactions between backbone constituents
Strong covalent bonds called disulfide bridges may reinforce the protein’s structure
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Fig. 5-21g
3 polypeptidesβ Chains
α Chains
CollagenHemoglobin
Quaternary structure results when two or more polypeptide chains form one macromolecule
- 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
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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
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Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008)
Formation of the 5’ Cap in mRNA
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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
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Figure 6-37 Molecular Biology of the Cell (© Garland Science 2008)
Formation of the 3’ PolyA tail in mRNA
The positionof the tail iscoded in DNA
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Figure 6-38 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
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Many proteins have alternative poly-A sites which caneither change the regulation of expression at the 3’UTRor, less commonly, change the length of the coding region.
The choice of poly-A sitecan be regulated by external signals
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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
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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
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Fig. 17-10
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5’ CapExon Intron5’
1 30 31 104
Exon Intron
105
Exon
146
3’Poly-A tail
Poly-A tail5’ Cap
5’ UTR 3’ UTR1 146
•RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence
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Examples of alternative RNA splicing (Part 1)
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Examples of alternative RNA splicing (Part 2)
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Alternative RNA splicing to form a family of rat α-tropomyosin proteins
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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!
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Dscam protein is required to keep dendrites from the same neuron from adhering to each other
Dscam complexityis essential to the establishment of the neural net by excludingself-synapses fromforming
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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 muscle-specific variants
- Trans-acting proteins recognize these sequences and recruit or block spliceosome formation at the site
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Muscle hypertrophy through mis-spliced myostatin mRNA
Splice sitemutationscan be verydeleterious,rarely can beadvantageous
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Fig. 17-11-1RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5�
Spliceosomesconsist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites
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Fig. 17-11-2RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5�
5�
Spliceosome
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Fig. 17-11-3RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5�
5�
Spliceosome
Spliceosomecomponents
Cut-outintronmRNA
Exon 1 Exon 25�
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Differential RNA Processing
Spliceosome proteins link directly to the nuclear pore to facilitate transfer of the spliced mRNA into the cytosol
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• 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
Alternative splicing can have very powerful effects on protein function
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Fig. 17-12Gene
DNAExon 1 Exon 2 Exon 3Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide
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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
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Cell type 1 Cell type 2
2. Cytosolic selection
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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
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Figure 6-40 Molecular Biology of the Cell (© Garland Science 2008)
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’ ‘translation ready’
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Key identifying proteins:Positive for export
cap and PolyA binding proteinsexon junction and SR proteinsnuclear export receptor
Negative for exportsnRNP
Positive for translationtranslation initiation factors
Negative for translationcap binding protein
The inappropriate combination of markers leads to degradation by nuclear exosome and cytosolic exonuclease
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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
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Figure 6-80 Molecular Biology of the Cell (© Garland Science 2008)
Eukaryotic block to translation
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Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008)
Prokaryotic block to translation
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3. Cell-Specific Regulation of Peptide and Protein Production
a. Regulation of translation
b. Co-/Post-translational protein regulation
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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
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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
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Figure 6-72 (part 1 of 5) Molecular Biology of the Cell (© Garland Science 2008)
a. The primary site of translation initiation is the 5’-cap
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Figure 6-72 (part 2 of 5) Molecular Biology of the Cell (© Garland Science 2008)
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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
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Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
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Fig. 17-10
Pre-mRNA
mRNA
Codingsegment
5’ CapExon Intron5’
1 30 31 104
Exon Intron
105
Exon
146
3’Poly-A tail
Poly-A tail5’ Cap
5’ UTR 3’ UTR1 146
c. 5’ caps and 3’ poly-A tails dictate the durationof time that the mRNA is active in the cytosol
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Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008)
c. 5’ caps and 3’ poly-A tails dictate the durationof time that the mRNA is active in the cytosol
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Figure 7-110 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 destroyedand/or
The 5’ cap is removed
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Figure 7-109 Molecular Biology of the Cell (© Garland Science 2008)
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2. External factors can extend RNA life
The length of translation can also respond to external regulation from hormones, growth factors, etc.
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Degradation of casein mRNA in the presence and absence of prolactin
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b. Co-/Post-translational protein regulation
1. Folding and membrane insertion
2. Covalent modifications
3. Polymer assembly
4. Proteolytic modifications
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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”
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Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)
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Fig. 5-24
Hollowcylinder
Cap
Chaperonin(fully assembled)
Polypeptide
Steps of ChaperoninAction:
An unfolded poly-peptide enters thecylinder from one end.
1
2 3The cap attaches, causing thecylinder to change shape insuch a way that it creates ahydrophilic environment forthe folding of the polypeptide.
The cap comesoff, and the properlyfolded protein isreleased.
Correctlyfoldedprotein
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Figure 12-43c Molecular Biology of the Cell (© Garland Science 2008)
Many membrane proteins are associated with the lipid bilayer during translation
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Figure 12-47 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)
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Figure Q12-5 Molecular Biology of the Cell (© Garland Science 2008)
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Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)
Misfolded proteins are controlled by regulated destruction
proteasome
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Figure 12-54 Molecular Biology of the Cell (© Garland Science 2008)
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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
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Figure 19-60b Molecular Biology of the Cell (© Garland Science 2008)
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Figure 3-27a Molecular Biology of the Cell (© Garland Science 2008)
3. Polymer Assembly
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Figure 19-62 Molecular Biology of the Cell (© Garland Science 2008)
42 genes in humans for α-collagen
You need three to make a protein
40 different proteins have been shown
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Figure 3-35 Molecular Biology of the Cell (© Garland Science 2008)
4. Proteolytic Modifications
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