7 - Genetic Transcription

7 - Genetic Transcription - 9/28/2008 Primers are added to...

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Unformatted text preview: 9/28/2008 Primers are added to the lagging strand in a 3’ to 5’ direction. Consequently, the Okazaki fragments are added in a 3’ to 5’ direction. Genetic Transcription and Translation Eileen Haase Fall 2008 – Lecture 7 Individual nucleotides (whether the RNA nucleotides in the primer or in the DNA) are added in a 5’ to 3’ direction (the direction of the arrows) 1 This primer is added first 2nd primer 1st DNA fragment 2nd fragment 2 Figure 5-7 Molecular Biology of the Cell (© Garland Science 2008) Direction of growth of chain Nucleotides are always added in a 5’ to 3’ direction DNA primase makes a primer out of ribonucleotides DNA polymerase makes DNA out of deoxy-nucleotides Direction of growth of primer 3 Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008) Central Dogma of Molecular Biology DNA RNA protein Replication: DNA DNA Transcription: DNA mRNA Translation: mRNA proteins (1o structure) 4 Next RNA added here Rules for Concepts/Vocabulary Key role of RNA: – mRNA - messenger RNA – tRNA - transfer RNA (adapter molecule) – rRNA - ribosomal RNA Protein & Nucleic Acid Synthesis There are a limited number of different monomeric building blocks: – 4 nucleotides: A, T (or U), G, C – 20 amino acids Vocabulary: Transcription, Translation Start/Stop/Regulation: comparison between Replication, Transcription & Translation 5 Monomers are added one at a time Each chain has a specific starting point. Growth proceeds in one direction: 5’ to 3’ There is a fixed and specific terminus (‘stop’). The primary synthetic product is often modified – DNA is packaged around + histones, RNA spliced, proteins are folded 6 1 9/28/2008 Processes in Molecular Biology – so far Polymer Process, Directionality 3 Roles for RNA Messenger RNA (mRNA) – copy of genetic information from DNA Subunit Enzymes Start Stop DNA Replication 5’3’ dNTPs DNA polymerase, ligase, etc. (repair) Origin of replication Entire chain Transfer RNA (tRNA) decodes mRNA into amino acids and protein sequence RNA (mRNA tRNA rRNA) Transcription 5’3’ NTPs (UTP not TTP) Ribosomal RNA (rRNA) - part of a large complex that physically move along an mRNA molecule, binds tRNA and catalyzes synthesis of proteins 7 8 protein Translation NC (amino carboxyl) Review: RNA vs DNA RNA Bases 5’ end Both DNA and RNA add nucleotides in a 5’ (phosphate) to 3’ (OH) direction 3’ end 9 RNA forms weak bonds and folds on itself - does not stay in a helix 10 Promoter is asymmetrical and only binds DNA in one direction: transcribes 5’3’ DNA is transcribed by RNA polymerase RNA polymerase opens double helix Provide energy Recognizes promoter Released after 10 nucleotides are made RNA polymerase does not use a primer and does not proofread (many more mistakes) RNA made from 5’ to 3’ 11 Bacterial transcription by RNA polymerase 12 2 9/28/2008 Videos 1001_RNA transcription Show QuickTime Movie (from ECB) of Essence of Transcription {Movies from L di h are d t il of {M i f Lodish details f eukaryotic/mammalian transcription} Bacterial transcription starts at a promoter on the DNA strand, and stops at a terminator Bacterial mRNA does not need processing – start making protein while the mRNA is still being transcribed. 13 14 Directions of transcription (bacteria) DNA does not match mRNA after processing. Eukaryotic mRNA has exons and introns RNA transcription always takes place in a 5’ to 3’ direction. The asymmetrical promoter orients the polymerase correctly. 15 Only 1% of DNA is used in most mammalian cells 16 Eucaryotic RNAs undergo processing in the nucleus Introns are removed (interfering) Exons remain 17 Genetic Organization: Prokaryotes vs Eukaryotes 18 3 9/28/2008 Eukaryotic mRNA: 5’ cap and poly A tail of several hundred nucleotides Remove introns Eucarytoic mRNAs have a 5’ cap, a poly A tail, and only one protein per gene. 5’cap found in eukaryotic mRNA Ready for cytoplasm! 19 20 Transcription DNA mRNA RNA polymerases link nucleotides together to form mRNA chain in 5` to 3` Many molecules of RNA polymerase can M l l f l simultaneously transcribe genes mRNA matures (splicing-introns removed) (splicingCompleted mRNA migrates from nucleus to cytoplasm 21 What is the “code” between DNA and amino acids There are four different nucleic acids (A, G, C, T), yet 20 different amino acids. How are the four bases ‘coded’ to get 20 possible amino acids? i id ? If each base coded a single amino acid, 41 = 4 possible amino acids If two bases are used to code each amino acid; 42 = 16 If three bases are used to code an amino acid; 43 = 64 22 Elucidating “Universal Code” 20 Amino Acids- 64 codons Genetic code is degenerate: There are many amino acids that have more than one codon (not a one-toone ratio between codons and amino acids) Test tube with synthetic mRNA, 20 different amino acids and protein building enzymes 23 24 4 9/28/2008 There is a lot of posttranscriptional processing: mRNA may be spliced before leaving the nucleus – remove introns The polypeptide chain may be modified before it becomes a functional protein 25 26 Transcription Translation Translation tRNA: •Chemically linked to a particular amino acid •Base-pair with a codon in mRNA 27 Translation: convert RNA codon to amino acid Cytoplasmic mRNA is ready to be used as the template for a protein Ribosome binds to mRNA at a start codon “AUG”, that is th t i recognized b i iti t tRNA i d by initiator Subsequent tRNAs bind to the ribosome and match the mRNA codon with the proper amino acid Release factor binds to one of 3 STOP codons (i.e. UAA, UAG, UGA), terminating translation and releasing the polypeptide 28 tRNA structure – due to H-bonds H- Codon Wobble The 1st and 2nd bases in an mRNA codon form WatsonCrick base pairs (A-U and C-G) with the 3rd and 2nd bases of a tRNA anticodon. 3rd mRNA base-pair may vary 20 < # tRNA < 61 There is more than 1 tRNA for some amino acids, and some tRNA molecules can base-pair with more than one codon. 30 2 regions crucial g to tRNA function One end of tRNA has an anticodon which binds with codon on mRNA, other end of tRNA has an amino acid. 29 Some tRNA recognize more than one codon Varies 5 9/28/2008 The genetic code – 3 nucleic acids represent an amino acid tRNA =attachment/translation/transfer function: ‘translating’ function: ‘translating’ mRNA into amino acid sequence Codon wobble in the third position helps to explain why so many of the codes for amino acids differ only in the third nucleotide. There are only 31 kinds of tRNA needed to fit the 20 amino acids to their 61 codons because of wobble base-pairings 31 Energy to make peptide bond 31 different tRNAs pair with 20 different synthetases for each of the 20 amino acids 32 AminoacylAminoacyltRNA synthetase attaches amino acids to tRNA Each synthase recognizes one amino acid and all the tRNAs that recognize codons for that amino acid Amino Acid + ATP + tRNA aminoacylaminoacyl-tRNA + AMP + 2Pi Takes energy to combine amino acid and tRNA Some of this energy is put into a high-energy highbond between the amino acid and tRNA. This bond supplies the energy for the peptide bond during protein elongation 20 different aminoacyl-tRNA synthases, one aminoacylfor each amino acid 33 34 Ribosome is the key enzyme in translation Huge ribosomal protein complex l binds mRNA, tRNA, and growing polypeptide Components of Eukaryotic Ribosome Lots of rRNA with proteins Two major subunits 35 36 6 9/28/2008 Sites on a ribosome Movies 0702_Prot Synthesis Show QuickTime Movie (from ECB) of Ribosome function during Translation Emphasis f E h i of GTP hydrolysis as ‘ l k’ f hd l i ‘clock’ for errorerror-checking. Ribosome holds 3 tRNAs and one mRNA 37 38 Protein Synthesis Participants mRNA – A copy of the DNA code for a specific protein (introns removed) The genetic code Aminoacylated tRNAs y – amino acid + tRNA + energy needed to make a peptide bond Ribosome – Where protein synthesis takes place – Aminoacylated tRNA and mRNA meet 39 40 Protein Synthesis Initiation – AUG – methionine – is the most common start codon – Initiation factors differ between eukaryotic and bacteria cells, but both require energy (GTP) Bacterial mRNA Elongation – elongation factors ( g g (EFs) ) – entry of aminoacyl-tRNA aminoacyl– formation of a peptide bond – movement of the ribosome one codon Termination – termination factors – UAA, UGA, UAG – stop codons – Cleave peptide chain from tRNA & release ribosomal subunits 41 Ribosome binds at “AUG” start codon and initiates protein synthesis. Can have multiple ribosomes on mRNA, all translating different proteins 42 7 9/28/2008 Initiation of Translation (eukaryotic) 5’ 3’ Translation -detail Amino end Sites: E-P-A 43 44 Elongation 1.Aminoacyl-tRNA binds to “A” site 2.Peptide bond between amino acids 3 and 4 causes release of amino acid from tRNA #3 3.tRNA #4 at “A” site of ribosome shifts forward to “P” site. tRNA #3 at “P” site shifts to “E” site. 4.tRNA #3 at “E” site is released, “A” site is open to next tRNA. 45 Stop codon Termination of protein synthesis Peptide release 46 Polyribosomes (polysomes) Have many ribosomes attached to mRNA, all translating proteins at different stages Processes in Molecular Biology Polymer Process, Directionality Subunit Enzymes Start Stop DNA RNA (mRNA tRNA rRNA) protein 47 48 8 9/28/2008 Processes in Molecular Biology Polymer Process, Directionality Central Dogma of Molecular Biology DNA->RNADNA->RNA->protein Subunit Enzymes Start Stop DNA Replication 5’ 5’3’ dNTPs DNA polymerase, ligase, etc. (repair) Origin of replication All cells, from bacteria to human express genetic human, information this way. Terminator RNA (mRNA tRNA rRNA) Transcription 5’ 5’3’ NTPs (UTP not TTP) RNA polymerase, etc. Promoter (bact: sigma factor) protein Translation NC (amino (amino carboxyl) aaaa-tRNA (amino acyl tRNA synthetase) Ribosome, etc. RibosomeRibosomebinding site Stop Codon 49 50 How do you prevent errors in translation? Codon is 3 nucleotides What if everything gets shifted by one nucleotide? – start ‘reading’ from nucleotide number 2 instead of number 1 Overlapping open reading frames – the problem of no commas between codons What if the mRNA is misread? – Miss a mucleotide – Read four at once 51 Stop codons terminate translation before a functional protein is produced 52 Eukaryotic: 5’ cap (prevents nucleases from breaking down mRNA before it is done being used as a transcript and poly A tail Eukaryotes vs Procaryotes Protein translation is much more complex in eukaryotes 53 54 9 9/28/2008 Control of Gene Expressions We have gone through the details of DNA RNA protein protein Control of Genetic Expression can take place at any or all of the following stages 1. Transcription of DNA into RNA 2. RNA processing 3. Translation 4. Protein Activity How is this process controlled? controlled? Most of these steps take energy, we don’t want to transcribe genes we don’t need 55 56 Control of Genetic Expression can take place at any or all of the following stages 1. Transcription of DNA into RNA 2. RNA processing 3. Translation 4. Protein Activity Transcriptional regulation Gene regulatory proteins bind to DNA via weak bonds 57 58 Gene Regulatory Protein motifs These three motifs are found in gene regulatory proteins in virtually all eucaryotic organisms, where they are responsible for controlling the expression of thousands of different genes. Each motif makes many contacts with DNA. homeodomain Lac Operon – an “ON” or “OFF” switch Glucose and lactose levels control the initiation of transcription of the lac operon – An operon is the gene + all the regulatory segments of the DNA (promoter, operator, etc) – Lac operon has all the enzymes needed to digest lactose Gene regulatory proteins can turn genes on (activators) or off (repressors) Leucine zipper Gene transcription can be switched on when [lactose] and [glucose] Gene transcription can be switched off when [lactose] 59 60 3 zinc fingers 10 9/28/2008 Lactose binds to its repressor to turn the lac operon on (repressor can’t bind to operator) Tryptophan binds to its repressor to turn its own gene off (repressor can bind to operator) 61 62 [Tryptophan] can turn genes off [Tryptophan] controls gene expression Repressor protein is inactive at low levels of tryptophan, gene to make tryptophan is turned ON High levels of tryptophan bind to repressor protein and activate it. Turn gene OFF When [tryptophan] , it is released from [tryptophan] the repressor protein and the gene is turned back on. 63 64 Genes expression doesn’t have to be digital Some genes are either ON or OFF Other genes can be expressed at low l l l levels or hi h l high levels The level of gene expression can be due to a number of factors Differing efficiencies of gene expression – ‘strength’ of promoter 65 66 11 9/28/2008 The production of a protein in a eukaryotic cell has many control points: Transcription control is most efficient 67 68 Questions? Key role of RNA: – mRNA - messenger RNA – tRNA - transfer RNA (adapter molecule) – rRNA - ribosomal RNA Vocabulary: Transcription, Translation Start/Stop/Regulation: comparison between DNA Replication, Transcription & Translation 69 12 ...
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