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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 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)