E. CELL SPECIALIZATION: RNA and Protein Regulation 1.nRNA to protein (review) 2.Cell-Specific...

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

Second mRNA baseF

irs

t m

RN

A b

as

e (

5

en

d o

f c

od

on

)

Th

ird

mR

NA

ba

se

(3

e

nd

of

co

do

n)

• 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

Fig. 17-13

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

Trp

Phe Gly

Codons 35

mRNA

Key Players in: 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

Amino acidsubunits

+H3N

Amino end

25

20

15

10

5

1

Primary 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

Fig. 5-21c

Secondar Structure

pleated sheet

helix

The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone

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

Fig. 5-21g

3 polypeptides Chains

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

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

Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008)

Formation of the 5’ Cap in mRNA

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

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


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

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

http://en.wikipedia.org/wiki/File:Alternative_polyadenylation.svg

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

Pre-mRNA

mRNA

Codingsegment

Introns cut out andexons spliced together

5’ Cap

Exon 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

Examples of alternative RNA splicing (Part 1)

Examples of alternative RNA splicing (Part 2)

Alternative RNA splicing to form a family of rat α-tropomyosin 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 complexityis essential to the establishment of the neural net by excludingself-synapses fromforming

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

Muscle hypertrophy through mis-spliced myostatin mRNA

Splice sitemutationscan be verydeleterious,rarely can beadvantageous

Fig. 17-11-1RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites


Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome


Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 25

Differential RNA Processing

Spliceosome proteins link directly to the nuclear pore to facilitate transfer of the spliced mRNA into the cytosol

• 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

Fig. 17-12

GeneDNA

Exon 1 Exon 2 Exon 3Intron Intron

Transcription

RNA processing

Translation

Domain 2

Domain 3

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

Cell type 1 Cell type 2

2. Cytosolic selection

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’ ‘translation ready’

Key identifying proteins:

Positive for exportcap 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

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


Prokaryotic block to translation

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

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

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

Fig. 17-10

Pre-mRNA

mRNA

Codingsegment

5’ Cap

Exon 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 duration of time that the mRNA is active in the cytosol


c. 5’ caps and 3’ poly-A tails dictate the duration of time that the mRNA is active in the cytosol


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

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”

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

Figure 12-43c Molecular Biology of the Cell (© Garland Science 2008)

Many membrane proteins are associated with the lipid bilayer during translation


Misfolded proteins are controlled by regulated destruction

proteasome

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


42 genes in humans for -collagen

You need three to make a protein

40 different proteins have been shown


4. Proteolytic Modifications

E. CELL SPECIALIZATION: RNA and Protein Regulation 1.nRNA to protein (review) 2.Cell-Specific...

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