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Transcript
___________________________________ 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 Ribosome Translation - mRNA - tRNA - ribosome - amino acids tRNA with amino acid attached ___________________________________ ___________________________________ ___________________________________ tRNA ___________________________________ Anticodon Codons 5 3 ___________________________________ mRNA ___________________________________ ___________________________________ ___________________________________ • 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 ___________________________________ 5 +H N 3 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 ___________________________________ ___________________________________ β pleated sheet ___________________________________ ___________________________________ α 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 ___________________________________ β Chains Quaternary structure results when two or more polypeptide chains form one macromolecule ___________________________________ ___________________________________ α 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 5 Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoprote ins (snRNPs) that recognize the splice sites RNA transcript (pre-mRNA) Exon 1 Intron Protein snRNA ___________________________________ Exon 2 Other proteins ___________________________________ snRNPs ___________________________________ ___________________________________ ___________________________________ ___________________________________ ___________________________________ Fig. 17-11-2 5 RNA transcript (pre-mRNA) Exon 1 Protein snRNA Intron ___________________________________ Exon 2 Other proteins ___________________________________ snRNPs Spliceosome ___________________________________ 5 ___________________________________ ___________________________________ ___________________________________ ___________________________________ Fig. 17-11-3 5 ___________________________________ RNA transcript (pre-mRNA) Exon 1 Intron Protein snRNA Exon 2 ___________________________________ Other proteins snRNPs Spliceosome ___________________________________ 5 ___________________________________ ___________________________________ Spliceosome components 5 mRNA Exon 1 Cut-out intron Exon 2 ___________________________________ ___________________________________ 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’ ‘translation ready’ Figure 6-40 Molecular Biology of the Cell (© Garland Science 2008) ___________________________________ ___________________________________ ___________________________________ 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 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. ___________________________________ ___________________________________ ___________________________________ ___________________________________ 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 α-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) ___________________________________
