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DNA Protein Mutations Reading Ch 17: Gene Expression Transcription Transcription • non-coding DNA - Promoters - Splicing: exons/introns • RNA structures Translation • tRNAs • Ribosome • splicing • ribozymes Homework Ch 48 Prequiz mutations can affect protein structure and function • Mutations are changes in the genetic material of a cell or virus • Point mutations are chemical changes in just one base pair of a gene • The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein How do mutations occur? Small changes can be devestating R = CH2CH2-COOH R = CH-(CH2)3 Types of Point Mutations • Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions More… Deletion The following would be a: 5’-C AUG CCU AGG UAG G-3’ Met-Pro- Arg stop 5’-C AUG GCU AGG UAG G-3’ Met-Asp - Arg stop a) Silent mutation b) Missense c) Nonsense d) frameshift Gene Structure Genes have a promoter, coding region and termination region Genes are more than code for protein Prokaryotic RNA polymerase binds promoters Initiation Terminator Termination point Coding Sequence Upstream of gene Downstream Gene A Gene X Gene Q Although no promoter shown, it is implied that there is one = Gene A Gene X Gene Q = Pro Gene X Promoter Gene X •Promoter indicates direction •Arrow indicated where transcription begins •ALL genes have a promoter Which strand is the template strand? a) Top b) Bottom c) Neither there is no template strand in transcription Synthesis of an RNA Transcript • The three stages of transcription: – Initiation (our focus) • melting and 1st nucleotide – Elongation – Termination RNA Polymerase •separates 2 strands and initiates transcription •No need for primer •Uses NTPs to start and build (polymerize) RNA Eukaryotic RNA Polymerase II Binding and Initiation of Transcription • Promoters signal the initiation of RNA synthesis Basal level • Transcription factors mediate the binding of RNA polymerase and the initiation of transcription • The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex • A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes – Fewer hydrogen bonds – easier to melt In eukaryotes transcription factors required Transcription bubble does NOT go both ways After RNA pol moves – DNA behind it renatures Nontemplate Elongation No need for: •Helicase •topoisomerase RNA polymerase 3’ RNA nucleotides 3’ end 5’ 5’ Newly made RNA Direction of transcription (“downstream”) Template Elongation of the RNA Strand • As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time • 40 nucleotides per second in eukaryotes • A gene can be transcribed simultaneously by several RNA polymerases • prokaryotes translation can occur simultaneously with transcription due to the lack of a nuclear membrane Termination • Bacteria: polymerase stops transcription at end of terminator • In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA AUG ppp- Translation start UAA -OH UAG UGA Translation stop Prokaryote gene/mRNA Structure Transcription Initiation Transcription Termination promoter DNA 5’ untranslated mRNA 5’-PPP 3’ untranslated AUG Translation Start Ribosome binding site Shine-Delgarno (only in bacteria) UAA Stop 3’-OH Eukaryotes: Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified: – The 5 end receives a modified nucleotide 5 cap – The 3 end gets a poly-A tail • These modifications share several functions: – They seem to facilitate the export of mRNA – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5 end RNA processing in Eukaryotes Eukaryote genes also have exons/introns Primary Transcript: or pre-mRNA After Transcription, before leaves nucleus Guanosine Cap added G-PPP Splicing components • snRNA – small nuclear RNA • snRNP – small nuclear ribonucleo-protein complex. Each has one snRNA and about 7 different proteins. • There are 5 different snRNPs, each with its own distinct snRNA (U1, U2, U4, U5, or U6) and distinct proteins. • Spliceosome – complex of all the snRNPs that work together to mediate splicing Biology Themes Reminder: Proteins do “work” / Proteins have “function” • ie – most tasks are completed by proteins • ex: G3P G3P dehydrogenase BPG Why are proteins functional? Biology Themes Reminder: Structure = Function • Proteins have diverse shape • The shape gives a unique (specific) pocket where interaction can occur • The active site is where specific electrons movement (chemical reactions) occur between catalyst and reactants Proteins can interact with DNA in a sequence specific manner But DNA is relatively uniform, so not always easy Protein DNA nucleotides Ribozymes • Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA • The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins • 3 properties of RNA enable it to function as enzyme – It can form a three-dimensional structure because of its ability to base pair with itself – Some bases in RNA contain functional groups – RNA may hydrogen-bond with other nucleic acid molecules RNA has many functional (reactive) groups 1 more OH (hydroxyl/alcohol group) compared to DNA Base Phosphate groups H Ribose Deoxyribose RNA has shape Shape = function like protein • Not as many as proteins though • • • Some areas are single stranded Some Double stranded (anti-parallel regions) DNA is mostly double-stranded (helix) and fewer options for shapes 1 unique aspect / advantage over protein is that because it is RNA it has a sequence. This allows it to bind other sequences with relative ease versus a protein, which a whole chain for a few interactions All of the rest of protein needs to have a complimentary For proteins shape to bind here snRNA acts like a linker snRNA binds mRNA and protein Proteins link to each other Only a set of proteins needed Rather than a different protein for every splice site as with restriction enzymes Loop lariat snRNAs are gene products DNA mRNA Protein NOT ALL GENES BECOME PROTEINS! The Functional and Evolutionary Importance of Introns • Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing • Such variations are called alternative RNA splicing • Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes Alternative splicing Domains • Proteins often have a modular architecture consisting of discrete regions called domains – can have a isolated function – Ex: DNA Pol I has a domain for exonuclease activity and a separate domain for polymerase activity • In many cases, different exons code for the different domains in a protein • Exon shuffling may result in the evolution of new proteins Domains • Proteins often have a modular architecture consisting of discrete regions called domains – can have a isolated function – Ex: DNA Pol I has a domain for exonuclease activity and a separate domain for polymerase activity • In many cases, different exons code for the different domains in a protein • Exon shuffling may result in the evolution of new proteins Translation: The Ribosome & transfer RNAs (tRNAs) The Ribosome • catalyzes peptide bond formation (joins amino acids) • Houses mRNA & tRNAs tRNAs determine what amino acid is used Carry correct amino acids to corresponding codon Intro Ribosome • Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis • The two ribosomal subunits rRNA gives ribosome shape Large subunit • 31 proteins • 2 rRNA 23S (~3000nt’s) 5S (~300 nt’s) Enzymatic Small subunit • 20 Proteins • rRNA 16S (~1500 nt’s) Forms bottom of A P E sites Where codons and anticodons interact Ribosomal proteins and rRNAs are encoded by genes rRNAs are NOT translated though Building a Polypeptide • The three stages of translation: – Initiation – Elongation – Termination • All three stages require protein “factors” that aid in the translation process Initiation 1. Prokaryotes: Shine-delgarno sequence orients small subunit just prior to AUG 1. Eukaryotes: small subunit binds to the 5’-cap • Translation starts at 1st AUG • AUG Methionine is always the first amino acid Initiation 2. Met-tRNA binds (tenuous) 3. Large subunit attaches (Takes Energy) Shine-Delgarno A ribosome has three binding sites for tRNA: –The P site holds the tRNA that carries the growing polypeptide chain –The A site holds the tRNA that carries the next amino acid to be added to the chain –The E site is the exit site, where discharged tRNAs leave the ribosome Elongation 1. Codon Recognition 3. Translocation 2. Peptide bond formation Termination of Translation • stop codon in A site • The A site accepts a protein called a release factor • causes the addition of a H2O instead of amino acid (Hydolysis) • This reaction releases the polypeptide, and the translation assembly then comes apart Ribosome 5’- C G A U G CCC AAA U A A C- 3' UAC E P Met A Initiation • Ribosome binds at shinedelgarno (prok) or 5’-cap (euk) • Translation starts at 1st AUG • AUG Methionine is always the first amino acid tRNA parts 3’ Phe • Many tRNAs 5’ • single-stranded RNA about 80 nucleotides long • Has 3D structure -Flattened looks like a cloverleaf • Amino acid attachment site • anti-codon that binds to the mRNA codon through base pairing anticodon A U G U U C U A A mRNA 5’- C G A U G CCC AAA UAC GGG UUU U A A C- 3' Release Factor Hydrolysis Met Pro Lys STOP Dehyration Synthesis / Condensation Reaction 5’- C G A U G CCC AAA U A A C- 3' Release Factor Met Pro Lys STOP Ribosome mRNA 5’- C G A U G CCC AAA U A A C- 3' UAC Met Each tRNA has a distinct anticodon sequence that will base-pair with a complementary codon on mRNA The methionine tRNA has an anticodon of UAC Ribosome 5’- C G A U G CCC UAC GGG Met Pro AAA U A A C- 3' Accurate translation requires a correct match between the tRNA anticodon and an mRNA codon Which of the following is an anticodon for Asn? a) UAA b) AUC c) UUA d) CGU Careful! Don’t use anticodon on the codon table Ribosome 5’- C G A U G CCC AAA UAC Met Technically 3’UAC5’ (antiparallel) so sometimes read as 5’CAU3’ U A A C- 3' Wobble: A single tRNA anticodon can be used for multiple codons • Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon A-C-? U-G-I Thr-tRNA Wobble: A single tRNA anticodon can be used for multiple codons • Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon A-C-U U-G-I Thr-tRNA Wobble: A single tRNA anticodon can be used for multiple codons • Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon A-C-C U-G-I Thr-tRNA Wobble: A single tRNA anticodon can be used for multiple codons • Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon A-C-A U-G-I Thr-tRNA tRNAs have shape 3’ 5’ RNA can be single-stranded OR double-stranded A hairpin loop Base pairing Hydrogen bonding tRNA have shape Structure = function some RNAs are ribozymes (RNA enzyme) ex: ribosomal RNA (rRNA) tRNA have shape tRNA shape and anticodon allows it to serve as a link / adapter between RNA and protein • anticodon allows it to interact with mRNA through base pairing – in a sequence specific manner • Shape allows proteins to bind tRNA more readily 5’- C G A U G CCC UAC GGG Met Pro AAA U A A C- 3' tRNAs are reused 5’- C G A U G CCC AAA U A A C- 3' UAC GGG Met Uncharged tRNA Pro UAC UAC Met aminoacyl tRNA synthetase Charged tRNA = aminoacyl tRNA Met tRNA have shape The tRNAs corresponding to a particular amino acid have the same shape that is distinct from others. example: •All Cysteine tRNAs have same shape •All phenylalanine tRNAs have the same shape •The shape of Cys tRNAs is different than the shape of Phe tRNAs Cys Phe Phe Translation • For Each codon 1 amino acid (ex: CCC is Pro) HOWEVER, • Code is redundant 1 aa has many codons (ex: Pro can be CCU, CCC, CCA CCG) Similar tRNAs are charged by the same tRNA synthetase Because tRNAs for the same amino acid have the same shape, they can all be recognized by the same aminoacyl-tRNA synthetase which will attach its amino acid ex: all theonine tRNAs will be recognized by threonine tRNA synthetase and have threonine attached to them 3 Ile tRNAs – anticodons: UAA, UAG, UAU How many shapes are there for these 3 Ile tRNAs? a) 1 b) 2 c) 3 d) 4 e) 5 How many aminoacyl tRNA synthetases are there for the 3 Ile tRNAs? a) 1 b) 2 c) 3 d) 4 e) 5 tRNA Structure • Accurate translation requires two steps: – a correct match between the tRNA anticodon and an mRNA codon – a correct match between a tRNA and an amino acid, attached by the enzyme aminoacyltRNA synthetase Ser anticodon A U G U U C U A A