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 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) 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 Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly Codons 3 5 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 acid subunits + H 3 N Amino end 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 Polypeptide backbone Hydrophobic interactions and van der Waals interactions Disulfide bridge Ionic bond Hydrogen bond 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 Collagen Hemoglobin 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 position of the tail is coded in DNA

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

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 Pre-mRNA mRNA Coding segment Introns cut out and exons spliced together 5’ Cap Exon Intron 5’ ExonIntron 105 Exon 146 3’ Poly-A tail 5’ Cap 5’ UTR3’ UTR 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 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 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 site mutations can be very deleterious, rarely can be advantageous

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoprote ins (snRNPs) that recognize the splice sites

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome components Cut-out intron mRNA Exon 1 Exon 2 5

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 Gene DNA Exon 1Exon 2 Exon 3 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 1Cell 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

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’

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

Figure 6-80 Molecular Biology of the Cell (© Garland Science 2008) Eukaryotic block to translation

Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008) 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

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 Molecular Biology of the Cell (© Garland Science 2008)

Fig Pre-mRNA mRNA Coding segment 5’ Cap Exon Intron 5’ ExonIntron 105 Exon 146 3’ Poly-A tail 5’ Cap 5’ UTR3’ 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) c. 5’ caps and 3’ poly-A tails dictate the duration of time that the mRNA is active in the cytosol

Figure 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 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 Hollow cylinder Cap Chaperonin (fully assembled) Polypeptide Steps of Chaperonin Action: An unfolded poly- peptide enters the cylinder from one end The cap attaches, causing the cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide. The cap comes off, and the properly folded protein is released. Correctly folded protein

Figure 12-43c Molecular Biology of the Cell (© Garland Science 2008) Many membrane proteins are associated with the lipid bilayer during translation

Figure (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Figure Q12-5 Molecular Biology of the Cell (© Garland Science 2008)

Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008) Misfolded proteins are controlled by regulated destruction proteasome

Figure 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)

Figure 3-27a Molecular Biology of the Cell (© Garland Science 2008) 3. Polymer Assembly

Figure 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 3-35 Molecular Biology of the Cell (© Garland Science 2008) 4. Proteolytic Modifications