Information Content in Genetics: DNA, RNA and protein mrna translation into protein (protein synthesis) Francis Crick, 1958 [Crick, F. H. C. in Symp. Soc. Exp. Biol., The Biological Replication of Macromolecules, XII, 138 (1958)] & 1970 [Francis Crick (1970) Central Dogma of Molecular Biology. Nature 227:561-563] proposed idea of Central Dogma that was revised later by inclusion of retrovirus reverse transcription from RNA to DNA and then back to RNA. 1
In prokaryotes DNA replication, DNA transcription into RNA and translation of mrna into proteins by ribosomes all occur in the cytoplasm. In eukaryotes DNA replication, DNA transcription into RNA, and RNA processing all occur in the nucleus and protein synthesis on ribosomes occurs in the cytoplasm. Eukaryotes have many more levels of regulation in the expression of the genetic code than do bacteria. https://www.mun.ca/biology/scarr/igen3_05-09_figure-l.jpg. jmp 13Sep2013 Eubacterial and eukaryotic protein synthesis have the same general pattern but there is more complexity in the eukaryote system. http://origin-ars.els-cdn.com/ content/image/1-s2.0- S0959440X09000700-sc1.jpg. jmp 11Sep2013 2
Prokaryote Protein Synthesis (mrna Translation) Initiation Initiation Factors IF1 and IF3 assist the 30S ribosomal subunit to align correctly with the mrna with its 5 Shine-Dalgarno sequence matching the 3 anti-shine- Dalgarno sequence of the 16S rrna. IF2 brings the formylmethionine-trna complex to pair with the mrna start codon AUG as IF3 falls off. Now the large 50S ribosomal subunit with its 23S and 5S rrnas can bind the small ribosome-mrna-fmet-trna complex in order for protein synthesis to proceed. http://www.nature.com/nature/journal/v475/n7354/ images_article/nature10126-f3.2.jpg. jmp 12Sep2013 http://bass.bio.uci.edu/~hudel/bs99a/ lecture23/hudel_27_18.jpg. jmp 11Sep2013 Prokaryote Protein Synthesis Elongation http://www.mun.ca/biology/ desmid/brian/biol2060/ BIOL2060-22/22_10.jpg. jmp 12Sep2013 3
There is a unique aminoacyl-trna synthetase for each amino acid-trna combination to ensure information integrity. See figure to the right. http:// canmedia.mcgrawhill.ca/istudy3/books/0070741751/images/figures/ bro41751_1115l_lg.jpg. jmp 12Sep2013 http://bioserv.fiu.edu/~walterm/genbio2004/ Chapter12_DNA_expression/figure%2012-07.jpg. jmp 12Sep2013 http://www.rcsb.org/pdb/education_discussion/ molecule_of_the_month/images/aars.gif. jmp 12SEp2013 The trna is the golden molecule to the left. Note the red spheres at the bottom representing the anticodon and the top red spheres where the amino acid phenylalanine is attached to the 3 CCA. http://cdn.physorg.com/ newman/gfx/news/hires/ 2009/31-researcherss.jpg. jmp 12Sep2013 http://www.rpi.edu/dept/bcbp/molbiochem/mbweb/ mb2/part1/images/protsyn.jpg. jmp 12Sep2013 Elongation Factor EF-G-GTP energized with GTP translocates the newly elongated Peptidyl-tRNA from the A (aminoacyl) site to the P (peptidyl) site as the old peptidyl trna leaves via the E (exit) site. As GTP hydrolyzes to GDP, EF-G falls from the A site and Elongation Factor Tu (EF-Tu) carrying a trna loaded with its amino acid positions the trna-amino acid in A site ensuring proper trna anticodon pairing with the mrna codon. The 23S rrna interacts with EF-Tu to catalyze the peptide bond between the new amino acid and the peptide chain hydrolyzing GTP to GDP in EF-Tu so that EF-Tu and its now empty trna leave the ribosome. http://www.pnas.org/content/106/4/1063/ F1.large.jpg. jmp 12Sep2013 http://www.sciencemag.org/content/335/6074/1366/ F2.large.jpg. jmp 12Sep2013 4
Paige S. Spencer & Jose M. Barral (2012) Computational Structural Biotechnology Journal, http:// dx.doi.org/10.5936/csbj.20 204006 image at http:// journals.sfu.ca/rncsb/index.php/csbj/article/viewfile/ 13/72/472. jmp 11Oct2013 Elongation Factor Tu (EF-Tu) carrying a trna loaded with its amino acid positions the trna-amino acid in the A site ensuring proper trna anticodon pairing with the mrna codon. Wobble position effects as well as relative amounts of a given trna-aa affect the rate at which the amino acid is positioned in the A site to carry out synthesis of the peptide bond between this new amino acid and the growing peptide in the P site. Many antibiotics interfere with bacterial protein synthesis. Besides treating infections, we use these antibiotics to study key reactions in protein synthesis. http://origin-ars.els-cdn.com/content/image/1-s2.0-s1074552110002048-gr1.jpg. jmp 11Sep2013 5
http://www.pnas.org/content/ 106/52/22239/F1.large.jpg. jmp 11Sep2013 The polypeptide is synthesized with its amino ( + NH3) end leading the way through the ribsome tunnel in the 50S subunit. Once free of the tunnel, the polypeptide begins to take its threedimensional shape forming domains. http://en.wikipedia.org/wiki/ File:Ribosome_mRNA_translation_en.svg. jmp 11Sep2013 http://www.nobelprize.org/educational/medicine/dna/ a/translation/pics/translation2.gif. jmp 12Sep2013 6
Prokaryote Protein Synthesis Termination Release Factor RF1 binds mrna codons UAA and UAG while RF2 binds mrna codons UAA and UGA mimicking a charged trna. They separate the peptide chain from the trna in the P site. Then RF3 which is a GTP-binding protein releases either RF1 or RF2 from the A site. Now Ribosome Recycling Factor along with Elongation Factor EF-G (also a GTP-binding protein) trigger release of RF3 and the trna in the Exit (E) site followed by the separation of the small 30S subunit from the large 50S subunit. IF3 finishes the disassembly process. http://origin-ars.els-cdn.com/content/image/1-s2.0- S0959440X07001984-gr1.jpg. jmp 12Sep2013 Eukaryote Protein Synthesis http://compbio.pbworks.com/f/central_dogma.jpg. jmp 10Sep2013 7
Eukaryote 80S ribosome large 60S and small 40S subunits from Jonathan Dinman s lab homepage http://dinmanlab.umd.edu/ Crystal_80S_sep_2012.jpg. jmp 12Sep2013 The small 40S ribosome subunit contains an 18S rrna. The blue and red colors show which proteins bind the rrna. The large 60S ribosome subunit contains 25S joined to 5.8S and 5S rrnas with red, gold and mauve colors with their respective proteins. An assembled eukaryote ribosome is 80S so 40S + 60S = 80S. In prokaryotes, the ribosome subunits 30S + 50S = 70S. Just what you learned in elementary math, right? nonlinear function The Svedberg unit is a coefficient for the sedimentation rate of particles in centrifuges. It is affected by particle mass, density and shape. Think of the rate of a feather falling and of a nickel falling. [Not in a vacuum, Larry] Which falls more quickly? Now if you glue the nickel to the feather, what happens to the rate of fall? http://www.nature.com/nrmicro/ journal/v9/n12/images/ nrmicro2655-f1.jpg. jmp 11Sep2013 Eukaryote Protein Synthesis Initiation There are six initiation steps with 8 or more protein factors that bind the initiator trna- Methionine complex to the small 40S ribosomal subunit (43S), followed by binding this new complex to the mrna (48S), which involves scanning the Kozak sequence, after which this complex is bound to the large 60S ribosomal subunit (80S). eif2 assists the initiator trna-met complex (methionine is not formylated) to bind to small ribosomal subunit with eif2b to reactivate eif2 by replacing its GDP with GTP. The 5' 7-methyl guanosine cap is recognized by eif4 (eukaryote Initiation Factor 4) and functions much like the Shine- Dalgarno sequence in prokaryotes to help position mrna on the initiator trna-met-40s ribosomal subunit for proper translation. Scanning occurs for the Kozak sequence [CC] RCCAUGG with R coding for A or G with A being more efficient to initiate translation at the start codon within the Kozak sequence. eif5 helps the large 60S ribosomal subunit bind to this complex. [eif6 has previously prevented precocious binding to the complex before the critical steps were completed. If a 3' polya tail is present on the mrna, it binds with the Poly(A)-binding protein and will fold back over the initiation complex binding to the 5' 7mG cap binding proteins eif-4g and eif-4e. http://www.mol.biol.ethz.ch/groups/ban_group/ Initiation/Initiation?hires. jmp 11Sep2013 8
http://www.nature.com/ scitable/content/ne0000/ ne0000/ne0000/ ne0000/14263719/ f4_jackson_nrm2838-f1.jpg. jmp 11Sep2013 http://www.nature.com/nrmicro/ journal/v9/n12/images/ nrmicro2655-f1.jpg. jmp 11Sep2013 http:// micro.magnet.fsu. edu/cells/ ribosomes/ images/ ribosomesfigure1. jpg. jmp 11Sep2013 Eukaryote Protein Synthesis Elongation The eukaryote elongation steps are very similar to those in the prokaryotes. The EF-G-GTP complex is EF2-GTP in eukaryotes. The protein elongation factor 1 alpha (EF-1α) is a key GTP-binding enzyme in protein synthesis which carries out the same function as the prokaryote EF-Tu. [Small groups of organisms in all eukaryote kingdoms use elongation factor-like protein (EFL) for the same purpose.] It brings the trna-aa complex to the large ribosome subunit Aminoacyl (A) site for the trna anticodon to pair with the mrna codon. Once pairing of complementary bases occurs, the EF-1α GTPase domain catalyzes GTP to GDP and EF-1α releases the trna-aa so that the amino acid can now form a peptide bond with the amino acid in the Peptidyl (P) site.] Eukaryote Protein Synthesis Termination The eukaryote release factor erf1 recognizes all three stop codons: UAA, UAG and UGA. Although it is structurally different from prokaryote RF1 and RF2 with one domain mimicking a trna with its anticodon, it has the conserved gly-gly-gln sequence needed to cleave the ester bond holding the polypeptide to the last trna in the P site. A second release factor erf3 functions much like prokaryote RF3 using GTP to help erf1 release the polypeptide and to cause the ribosomal subunits to dissociate and fall away from the mrna. 9
Thomas Becker et al (2011) Nature Structural & Molecular Biology 18:715-720 http:// www.nature.com/nsmb/journal/v18/n6/images/ nsmb.2057-f4.jpg. jmp 12Sep2013 Release factors in Eukaryote Protein Synthesis Termination http://www.nature.com/embor/journal/v11/n3/images/embor201025-f1.jpg. jmp 12Sep2013 http://www.unifr.ch/biochem/ assets/images/dreyer/ research/sirna/image001.gif. jmp 12Sep2013 silencing RNAs (sirna) and micro RNAs (mirna) regulate DNA transcription and protein synthesis http://www.nature.com/npp/journal/v38/n1/ images/npp201287f1.jpg. jmp 12Sep2013 http://www.nature.com/srep/ 2012/121218/srep00996/images/ srep00996-f7.jpg. jmp 12SEp2013 10
Here they are using the bacterial protein elongation factor EF-Tu in the eukaryotic ribosome system to study similarities of binding. http://origin-ars.els- cdn.com/content/image/1- s2.0-s0959440x12001145- gr1.jpg. jmp 11Sep2013 http://rna.ucsc.edu/rnacenter/images/ 70s_atrna.jpg. jmp 11Sep2013 11
http://employees.csbsju.edu/ hjakubowski/classes/ch331/ dna/genessimplecomplex.gif. jmp 23Aug2013 Hierarchy of protein structure -- primary, secondary, tertiary, and sometimes quaternary Primary: linear order of amino acids with their distinctive R groups. Secondary: α helices and β pleated sheets held together by Hydrogen bonding. Tertiary: salt (ionic) bonds and disulfide (covalent) bonds plus H bonds and hydrophobic interactions (van der Waals forces) along with water partitioning help hold the polypeptide in its 3-Dimensional shape. Quaternary: two or more polypeptides associate to form a functional protein. 12
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http://www.icelandicsheep.com/ SheepMasterListPics/SRX534K-AI.jpg. August 29, 2006 http://www.jpmoth.org/ Bombycidae/Bombycinae/ L1Bombyx_mori.jpg. August 29, 2006 http:// instruct1.cit.cornell.edu/ courses/biog105/pages/ demos/105/unit1/media/ fibroin.jpg. August 29, 2006 14
Curly hair, perms and disulfide bonds http:// www.rahmqvist.co.uk/ school_scissors.aspx. 1Sep2010. 15
Enzymes (catalytic proteins) -- lysozyme, amylase, catalase, trypsin, polymerases, etc http://www.mol.biol.ethz.ch/groups/ glockshuber_group/research/tma/ image. August 29, 2006. amylase Domains are regions of specific function within a given polypeptide. Active site is where chemical reaction is catalyzed. 16
Francis Crick, 1958 [Crick, F. H. C. in Symp. Soc. Exp. Biol., The Biological Replication of Macromolecules, XII, 138 (1958)] & 1970 [Francis Crick (1970) Central Dogma of Molecular Biology. Nature 227:561-563] proposed idea of Central Dogma that was revised later by inclusion of retrovirus reverse transcription from RNA to DNA and then back to RNA. 17