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Biostatistics Week 5 Notes

by: Kiara Lynch

Biostatistics Week 5 Notes BIO 472

Marketplace > La Salle University > Biology > BIO 472 > Biostatistics Week 5 Notes
Kiara Lynch
La Salle

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This 10 page Class Notes was uploaded by Kiara Lynch on Monday February 22, 2016. The Class Notes belongs to BIO 472 at La Salle University taught by in Summer 2015. Since its upload, it has received 30 views. For similar materials see Biostatistics in Biology at La Salle University.


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Date Created: 02/22/16
Molecular Week 5 Notes (2/15-2/19) TRANSCRIPTION  Initiation of transcription o Promotor contains the TATA box which is 25 nucleotides away from the start site of transcription o Through TBP, TFIID recognizes and binds the TATA box  Enables adjacent binding of TFIIB  TBP- TATA binding protein  Single chain folded into two similar domains  Subunit of general transcript factor TFIID that is responsible for recognizing and binding to the TATA box sequence in DNA o DNA distortion- causes a unique DNA bending  2 kinks in double helix separated by partly unwound DNA o Rest of transcription factors and the polymerase assemble at the promotor o TFIIH- helicase and kinase activity  Binds after TFIID binds to TATA box  Pries apart DNA and uses ATP to phosphorylate the serines in the CTD near the new RNA transcript entry site  CTD- C-terminal domain; 52 repeats of a 7 amino acid sequence containing many serines; site of transcription  Uses ATP to pry apart DNA at start point which exposes the DNA template  Phosphorylates RNA polymerase (allosteric)  conformational change  polymerase released from general factors and elongation can begin o Conformational changes  Phosphorylated polymerase; rutter; clamp  Site of phosphorylation- long C terminal polypeptide tail (CTD)  Mediator protein (eukaryotic cells) o Gene regulating proteins help RNA polymerase, TF, and mediator assemble at the promotor o Activators attract ATP-dependent chromosome remodeling complex and histone acetylases o Binds to DNA far away- enhancer protein o Activates start of transcription o Binds to specific sequence- enhancer sites o DNA flexibility- also a mediator binding site o Mediator brings cofactors- chromosome remodeling complex or enzymes to modify histone tails  Gene to protein- Eukaryotes vs prokaryotes o Prokaryotes  mRNA is ready for translation- no further modifications necessary  5’ end of mRNA is produced by initiation of transcription  3’ end is produced by termination of transcription  No nucleus transcription and translation occur in a compartment  Translation of bacterial RNA usually begins before synthesis is completed  Instructions for multiple proteins o Eukaryotes  Stuck in nucleus  Primary RNA transcript is transcribed with introns which need to be removed so exons can link  splicing  Human beta-globin gene encodes 1 of the subunits of the oxygen carrying protein hemoglobin (has 3 exons)  Larger human Factor VIII gene is larger and has 26 exons and codes for protein that functions in the blood-clotting pathway; mutations lead to hemophilia  Modification to both ends  5’ cap o G residue covalently linked via triphosphate bridge in 5’ to 5’ linkage; b/w 7-methyl G residue and 5’ end of RNA transcript o Phosphorylated serines attract proteins for capping, adding poly A tail, and splicing o Phosphatase- remove P from 5’ end o Guanyl transferase- adds GTP  knocks off 2 phosphates and adds a 4rd phosphate o Methylase- methylates G residue  Poly A tail (200-250 A residues to 3’ end) o Gets out of nucleus; makes recognizable by ribosome; fuse/lifespan depends on it  loss of some A residues every time it is translated  Ends modified, introns removed by splicing, mRNA from nucleus to cytoplasm  Information for 1 protein; no introns  Intron removal strategy o 1 level  Pre-mRNA splicing reaction- adenine in intron sequence attacks 5’ splice sites and cuts backbone of RNA  5’ end covalently links to adenine creating a loop  3’ OH end of exon sequence joining two exons together and releasing intron sequence (lariat structure, eventually degraded)  Forms a continuous coding sequence; 3’ to 5’ exon linking  Signals in transcript to tell where junction point is  A residue surrounded by nucleotides is activated- needs to be able to participate in breakage of backbone of exon intron entry  Alternative splicing- same gene can produce different proteins  Ex: tropomyosin can produce striated muscle, smooth muscle, and fibroblast and brain mRNA o Coiled coil protein that regulates contraction in muscle cells o Not all exons are used in all transcripts  Consensus nucleotide sequence- specific sequences; 3 blocks of nucleotide sequence are needed to remove an intron sequence  RNA molecules can hybridize  R=A/G, Y=C/U o 2 ndlevel  RNA splicing catalyzed by assembly of snRNPs and other proteins, which together, constitute spliceosome  Spliceosome recognizes the splicing signals on a preRNA molecule, brings 2 ends of intron together, and provides enzyme activity for the 2 reaction steps  1. U1, snRNP, BBP, and U2AF recognize the branch point site (A)  2. U2 snRNP replaces BBP and U2AF and base pairs with branch point site consensus sequence  3. U4/U6 replaces U1; activates A for phosphoryl transferase rxn  RNA molecules act like enzymes  snRNP small nuclear ribonuclear proteins- combo of RNA and protein  4. U4/U6 base pairs break apart and U6 displaces U1 at splice nd junction to form active site for 2 phosphoryl transferase rxn (completes splice); Proteins associated with snRNPs are ATPases  Hydrolysis of ATP allows them to rearrange  5. Excised intron sequence in the form of a lariat (degraded in nucleus, snRNPs recycled) o 3 level  Exchange of U1 snRNP occurs before the first phosphoryl transfer reaction  Requires 5’ splice site to be read by 2 different snRNPs which increases the accuracty of 5’ splice site selection by the spliceosome  Branch point site is first recognized by BBP and subsequently by U2 snRNP  Check and recheck  increased accuracy for selection  Binding of U2 to branch point forces adenine to be unpaired and activates it for the attack on the 5’ splice site  Skip AU to CG- nothing for A to base pair with  squeezes and changes shape of backbone; activates A  Taking substrate and putting stress on bond (RNA acting like enzyme) o Formation of Lariat  2 exons brought in close proximity for the second phosphoryl transferase reaction  snRNPs position reactants and provide catalytic sites for the 2 rxns  U5 snRNP is present in spliceosome before rearrangement occurs  All the RNA components bring substrates together for proper base pairing  Need additional proteins and ATP hydrolysis  RNA acts like enzyme when it brings activated A next to splice site (bringing 2 substrates together) o Possible mistakes that can occur  Exon skipping  One of the sequences gets missed  Cryptic splice-site selection  Cryptic splicing signals are nucleotide sequences of RNA that closely resemble true splicing signals  Introduce splice site that didn’t exist before  Variation in exon and intron lengths o Average exon length- about 150 base pairs  Exon length is much more uniform o Much more consistent to find exons than look for introns o More size variation in introns  Identifying introns over exons o SR proteins bind to each sequence in pre-mRNA and help guide snRNPs to proper intron/exon boundaries  SR proteins- contain a lot of serines and arginines  Introns in mRNA are packaged into hnRNP complexes o Done during transcription; uniform exons o Bind to specific sequences within exons o CBC- cap binding complex  Mutations (splicing issues) in beta-globin gene (3 variations) o Normal adult beta-globin primary RNA transcript- normal mRNA formed from 3 exons o Some single-nucleotide changes that destroy a normal splice site cause exon skipping- mRNA with exon 2 msising o Some single-nucleotide changes that destroy normal splice sites activate cryptic splice sites- mRNA with extended exon 3 o Some single-nucleotide changes that create new splice sites that cause new exons to be incorporated- mRNA with extra exon inserted between exon 2 and exon 3  Generating 3’ end of eukaryotic RNA o Consensus nucleotide sequence- directs cleavage and polyadenylation to form 3’ end of eukaryotic RNA  CA bound by 3 factor; hexamer bound bg; CPSF- GU or U rich region bound by CstF o More complicated than in prokaryotes in which transcription stops at a termination signal and releases both 3’ end of transcript and the DNA template o Cleavage and poly-A signals encoded in DNA; cleavage factors released, RNA cleaved; RNA polymerase (PAP) in; RNA polymerase release, eventually terminates; poly-A binding protein in (CPSF); poly-A length regulation (adding phosphates)  Export ready mRNA molecule and transport through nuclear pore o Some proteins travel with the mRNA as it moves through the pore, others remain in the nucleus o Gate keepers making sure changes have been made o Initiation factors for protein synthesis help with translation later on; will only be present if there is a 5’ cap and a poly-A tail o Nuclear export receptor- complex that is deposited when mRNA is correctly spliced and polyadenylated o mRNA exported to cytosol; receptor dissociation from mRNA and is reimported into nucleus (reused) o final check- “nonsense-mediated decay”  after it leaves nucleus and before it loses the CBC  after check, mRNA continues to shed previously bound proteins and acquire new ones before it is efficiently translated into protein  rRNA- ribosomal RNAs o produced as 1 giant transcript  post translational modifications  cleaved into individual components that get transported out o S value- rate of sedimentation in an ultracentrifuge; the larger the S value, the larger the mRNA o 2 modifications of precursor mRNA by guide RNAs  Pseuoduridine- isomer of uridine (NH group)  2’-O-methlyated nucleotide- CH3 group o In nucleolus  snoRNAs (small nuclear RNA) determine sites of modification by base pairing to complementary sequences on precursor rRNA  snoRNAs bound to proteins and complexes are called snoRNPs o contain both guide sequence and enzyme that modify the rRNA TRANSLATION  3 reading frames in protein synthesis o Ribosome needs to get the correct reading frame by identifying where to start translation (at the Methionine) o Reads 5’ to 3’ in sets of 3 nucleotides o Only 1 reading frame contains the actual message  tRNA- transfer RNA o Single strand that folds because of internal base pairing (clover leaf shape) o Anticodon determines which amino acid gets added- 3 nucleotide sequences that base pair with a codon in mRNA o Amino acids are added to the growing polypeptide chain o 7-90 nucleotides in length; each one carries a covalently linked amino acid o 3 stop codons; 64 codons total o L shaped molecule- all tRNAs have a similar structure (CGCA)  Wobble base pairing between codons and anticodons rd o If nucleotide in first column is present at the wobble (3 ) position of the codon, it can base pair with any of the nucleotides in the second column  Prokaryotes Eukaryotes  U A, G, I U A, G, I  C G, I C G, I  A U, I A U  G C, U G C  Unusual nucleotides in tRNA o Produced by covalent modifications after incorporation into RNA chain o 10% of nucleotides are modified  tRNA splicing endonuclease o Docked to precursor tRNA o Endonuclease (4 subunit enzyme) o Removes tRNA introns o A 2ndenzyme, a multifunctional tRNA ligase, joins the two tRNA halves together  Peptidyl transferase o One known, all-RNA enzyme (no protein) o Amino acid activation o tRNA synthetases- different tRNA for each amino acid o Aminoacyl-tRNA synthetase enzyme; amino acid attaches to tRNA via ATP hydrolysis; amino acid is activated by binding with an AMP moiety  forms an adenylated amino acid on 3’ end of tRNA  forms aminoacyl-tRNA o Amino acid in active site of ATP synthase- hydrolyzed  AMP covalently linked to carboxyl side  bond broken  amino acid transferred to 3’ end of tRNA and is covalently linked o Aminoacyl-tRNA linkage  Carboxyl end of amino acid forms an ester bond with the ribose  Hydrolysis of the ester bond leads to a large favorable change in free energy which activates the amino acid  2 major classes of synthetase enzyme  Link amino acid directly to 3’ OH  Links initially to 2’ OH; transesterification shifts amino acids to 3’ position  Genetic code translated by 2 adaptors o tRNA synthetase- makes sure the correct amino acid attaches to tRNA o 1. Aminoacyl tRNA- couples amino acid to correct tRNA o 2. tRNA- anticodon base pairs with appropriate codon on mRNA o If there is an error in either step, the wrong amino acid will be added  Hydrolytic editing- way in which tRNA synthetases remove their own coupling errors of incorrectly attached amino acids o tRNA synthetase  Looks like DNA polymerase  Has 2 active sties (adding and editing)  Editing site- fits similarly shaped amino acids but correct amino acid is rejected and will not fit into the editing site o DNA polymerase error correction process  Removal process depends strongly on mispairing with the template  Has both polymerizing and editing site  tRNA synthetase and DNA polymerase are evolutionarily related  Recognition of tRNA by its aminoacyl tRNA o Correct tRNA in correct synthetase o Correct amino acid on correct tRNA o Specific nucleotides in anticodon and amino acid accepting arm allow correct tRNA to be recognized by the synthetase enzyme o tRNA, editing site, tRNA synthetase, ATP that helps link the amino acid  Incorporation of amino acids into a protein (form polypeptide) o Stepwise addition to C-terminal end o Peptide bond formation is energetically favorable because the C terminus is activated by covalent attachment of tRNA o Peptidyl tRNA attached to C-terminus of the growing polypeptide chain; in specific spot in ribosome o Aminoacyl-tRNA- has amino acid attached; enters ribosome and a peptide bond is formed; becomes new peptidyl tRNA o New peptidyl tRNA molecule attached to C-terminus of the growing chain  Ribosomes- protein synthesis o In cytoplasm- free or attached to membrane of endoplasmic reticulum  S value- how far they move in a cesium chloride gradient o Eukaryotic  Larger protein components of subunits- keeps stable  RNA does most of the work  Form active site  Position properly  Catalyze reactions through H bonding o Prokaryotic  Nearly same structure; functions similarly  RNA binding sites in ribosomes o 3 binding sites formed between large and small subunits o 1 tRNA binding site o 3 tRNA binding sites  E site- exiting; where peptide tRNA leaves  P site- peptide tRNA most often found here  A site- aminoacyl tRNA most often found here o Core is mostly RNA molecules (mRNA)  Translation o Add 1 amino acid to existing polypeptide chain o Amino acid selected by complementary base pairing with anticodon on its attached tRNA molecule and next codon in mRNA chain o Step 1- aminoacyl tRNA molecule binds to vacant A site on ribosome  spent tRNA molecule dissociates from A site  tRNA 2 in exiting site; tRNA in aminoacyl site; new AA brought in by tRNA o Step 2-new peptide bond formed o Step 3- large subunit translocates relative to small subunit, leaving 2 tRNas in hybrid sites  Movement of large subunit  ½ site (E site) in tact  2 hybrid sites (E/P and P/A)  Uses energy from the peptide bond to move o Step 4- small subunit translocates carrying its mRNA a distance of 3 nucleotides through the ribosome  “resets” ribosome (empty A site)  Reform 3 sites, peptidyl tRNA in P site, amino acid brought in, open A site  Proofreading o Makes sure correct amino acid is in place o EF-tu- multidomain GTPase  Provides 2 opportunities for proofreading of the codon-anticodon match  Incorrectly paired tRNAs are selectively rejected which increases accuracy o EF-tu with GTP bound binds to synthetase  incorrectly base-paired tRNAs preferentially dissociate  P released  GDP released  incorrectly base-paired tRNAs preferentially dissociate o EF-G- GTPase elongation factor  To move small subunit  Binds to half site  Conformational changes from GTP hydrolysis cause translocation of small subunit  Reconnection of 3 sites o Recognition of correct codon-anticodon matches by small subunit rRNA of ribosome  Information is transmitted via H bonds and 16S RNA to active site of EF-tu which leads to conformational changes  Ribosome o Core is mostly RNA o L1 subunit- characteristic protrusion; binds to outside and helps to stabilize conformational changes taking place o 6 domains  continuous o Individual subunits base pair internally  Initiation of protein synthesis in eukaryotes o eIF4E binds to 5’ cap o Requires poly A tail of mRNA bound by poly A binding proteins which interact with eIF4G o Makes sure both ends of mRNA are intact before initiating protein synthesis o Initiator tRNa- tRNa for methionine with Met covalently linked by Met and RNA synthetase  Amino acid covered by GTPase like protein (EF-tu)  Looks for fully formed RNAs, binds to central cav; ½ P site o Initiator tRNA moves along RNA searching for first AUG (ATP hydrolysis) o eIF2 and other initiation factors dissociate o Large ribosomal subunit binds  Base pairing between codon and anticodon  info transmitted to initiation factor via GTP hydrolysis; initiation factor dissociates which makes room for the large subunit  Bacterial mRNA molecule o Single message produces different proteins based on where synthesis starts o Initiate transcription at ribosome binding sites o Permits backbone to synthesize more than 1 type of protein from a single mRNA molecule  Final phase of protein synthesis o 3 stop codons- don’t bind tRNA, bind release factors o Binding of release factor to A site bearing a stop codon terminates translation o Nothing to hold ribosome together- dissociates into its 2 subunits via GTP hydrolysis o Completed polypeptide is released  Polyribosome o Opportunity for amplification- can simultaneously translate the same eukaryotic gene (mRNA molecule) and produce multiple RNAs  Translational recoding o Serine modified  selenocysteine- looks like cysteine but instead of sulfur there is a cesium o Stop codon to new tRNA binding site o Incorporation of selenocysteine into growing peptide chain o Special tRNA charged with serine by seryl-tRNA synthetase o Serine converted to selenocysteine o Specific RNA structure in mRNA (stem and loop) signals for selenocysteine to be inserted in neighboring UGA codon  Selenocysteine-specific translation factor- GTPase- does the same thing as EF-tu but binds to downstream signal of mRNA; tells tRNA to bind and insert selenocysteine  Serine + seryl-tRNA synthetase  serine enzymatically converted to selenocysteine  selenocysteine-specific translation factor binds  signal that preceding UGA encodes selenocysteine  selenocysteine added to growing peptide chain  Proofreading – frameshifting that produces reverse transcriptase and integrase of a retrovirus o Improper mRNA splicing  Introns are not all taken out  Makes different protein or produces a stop codon o If an intron is left in stop codon- test ribosome stops and all downstream exong junctions remain intact  signals degredation o Exon junction complex- signals 2 exons have joined- stay with mRNA as it leaves nucleus o Test translation- grabs 5’ end and attempts to synthesize protein  comes in contact with exon junction  removes proteins  translation o Proteolytic processing of large protein consisting of both Gag and Pol amino acid sequences o Processing of more abundant Gag proteins produces viral capsid proteins o Gag and Gag-Pol fusion proteins start with identical mRNA  Gag protein terminates at stop codon downstream  Gag-Pol protein bypasses stop codon  Made possible by a controlled translational frameshift  tRNA attached to C terminus of peptide chain slips backward by 1 nucleotide on ribosome  pairs with UUU instead of UUA (incorporation codon)  next codon AGG in new reading frame specifies R instead of G


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