Week 3 Notes: Transcription and Translation
Week 3 Notes: Transcription and Translation BIOL 3510
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This 5 page Class Notes was uploaded by Marin Young on Wednesday February 3, 2016. The Class Notes belongs to BIOL 3510 at University of North Texas taught by Dr. Chapman in Spring 2016. Since its upload, it has received 52 views. For similar materials see Cell Biology in Biological Sciences at University of North Texas.
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Date Created: 02/03/16
Week 3: Chapter 7 | BIOL 3510 Notes by Marin Young RNA and the Cell: • RNA has an extensive range of types and functions because it's single-stranded and can fold on itself in many ways ○ Imagine a long strip of Velcro, only with alternating soft and rough pieces every inch or so--how many ways could you attach various little sections to create different structures? What if the alternating sections were different lengths and had to match up perfectly? Unconventional hydrogen-bonding: imperfect matches (like two hydrogen bonds between A and G even though G wants three) stabilize RNA structures □ Why not perfect match? If the RNA holds its shape weakly, it can be dynamic like a protein ○ RNAs can serve catalytic purposes ○ Recall structural differences from DNA: ribose instead of deoxyribose (extra oxygen on 2' carbon), and uracil replaces thymine to base- pair with adenine • Types of RNA: ○ Ribosomal rRNA is encoded by the DNA genome and transcribed in the nucleolus RNA polymerase I transcribes rRNA Ribosomal proteins enter the nucleolus to be assembled into ribosomes (rRNA and proteins) ○ Messenger mRNA is translated into proteins by ribosomes RNA pol II transcribes mRNA ○ Transfer tRNA is literally t-shaped and carries an amino acid and an anticodon to a ribosome RNA pol III transcribes tRNA The anticodon is complementary to the mRNA codon, and the amino acid is in position to bind to the last amino acid Major example of structure determining function--tRNA has regions (like protein domains) with functions for grabbing a specific amino acid (this is really impressive) and binding to the ribosome and stuff ○ Short nuclear snRNAs are important in mRNA splicing--example of catalytic RNA (sometimes called ribozymes) ○ 7sRNA and long non-coding lncRNA are both involved in regulation Briefly mentioned as examples of types of RNA--just be familiar with them Transcription of DNA to mRNA: • RNA pol II (or just RNA pol in prokaryotes) transcribes DNA to mRNA and depends on transcription factors ○ Transcription factors make up a third of the genome ○ Accessory proteins cause RNA polymerase to bind and leave at the appropriate places Bacteria have only one type of accessory protein, the sigma (σ) factor □ The sigma factor binds to two mini promoter regions (specific DNA sequences just ahead of the gene) and positionsRNA polymerase □ Sigma factor leaves once RNA pol starts moving down the DNA □ Termination (control of where RNA polymerase stops transcribing) can happen with or without a protein Accessory proteins often bind to a TATA box, a sequence of TATATATATA… □ Often found in the bacterial promoter region around position-10 ○ RNA polymerases have structural features with important functions An entry channel lets NTPs (nucleoside triphosphates, like ATP) into the active site A cytoplasmic tail domain (CTD) gets phosphorylated to activate the polymerase • Transcription in eukaryotes is complicated by nucleosome structure: the transcription complex has to be organized, oriented, and activated ○ For this reason, there are more specialized transcription factors for eukaryotes ○ TFII_s (Transcription Factors for RNA polymerase II, distinguished with letters A-H) are used for mRNA synthesis TFIID contains TBP, the TATA-binding protein □ Most of these abbreviations really do make sense--if they give you a hard time, look for a glossary of □ Most of these abbreviations really do make sense--if they give you a hard time, look for a glossary of acronyms in my exam 1 study guide □ Binding to the TATA box distorts the double helix and helps other TFs (TFIIB, but that's probably not too important) bind to the DNA □ TFIID also recognizes RNA polymerase, so it helps RNA polymerase bind near the TATA box TFIIH phosphorylates the CTD to activate the polymerase Other TFs bind to regulatory DNA sequences, stabilize unwrapped (unwrapped) DNA, and slide nucleosomes (like the chromatin remodeling complex) ○ RNA polymerase moves 3' to 5' on the template strand to make mRNA 5' to 3' (just like the nontemplate strand) Each nucleotide added is an NTP (a nucleoside triphosphate), which provides the energy to make the new phosphodiester bonds between nucleotides □ The nucleoside triphosphate gets hydrolyzed to give a nucleotide (attached to the one before it) and a molecule of pyrophosphate □ Bond forms between phosphate and 3' -OH □ Pyrophosphatase cleaves pyrophosphate to give two "inorganic phosphates" (orthophosphate / Pi/ PO 4- This provides a lot of energy ○ Several transcription complexes can transcribe the same gene simultaneously--as soon as one transcription complex is far enough away, transcription factors can bind to the TATA box, bind another RNA polymerase, and start transcribing In bacteria, a ribosome can even come start translating a segment of mRNA before all of it has even been transcribed, since prokaryotes don't spatially separate transcription from translation Since eukaryotes transcribe in the nucleus and translate in the cytoplasm or ER,transcription and translation are spatially and chronologically separated (different place, different time) • The mRNA transcript must be modified before it leaves the nucleus ○ Splicing: introns are removed, exons are spliced (combined) together A spliceosome (literally "splicing body") is made of catalytic snRNA (which acts as an enzyme to help break and form bonds in the mRNA) and SNRPs (snRNA-binding proteins) Two substitution reactions are involved in connecting two exons and freeing an intron lariat (lasso shape) □ A nucleotide's 2' -OH (the one not present in deoxyribose) substitutes with the 5' end of the intron, and then the 3' end of the first exon substitutes with the 3' end of the intron--I couldn't tell you whether to expect this level of detail on a test, but knowing the picture is probably best ○ Eukaryotic mRNA also needs a 5' cap and 3' poly-A tail to protect it from being degraded in the cytoplasm ○ Introns were once considered pointless "junk DNA," but their likely purposes include spacing out genes, helping regulate transcription, and, most important,alternative splicing Sometimes exons can be removed, reordered, or even trimmed during mRNA splicing, based on different regulatory short RNAs expressed in different cell types Two examples to know: human -tropomyosin and Titin (a giant muscle protein) both exhibit lots of alternative splicing •The 5' cap has a methylated guanosine attached by a triphosphate bridge. Image from Wikimedia Commons. •Each side of the gene transcript has an untranslated region (5' and 3' UTRs), about 50 nucleotides long •The 3' end is polyadenylated: it literally just has 150-250 adenosine nucleotides after the UTR •1st arrow indicates a start/Met codon; 2nd arrow indicates stop codon The proteins responsible for splicing, capping, and polyadenylation bind to RNA polymerase II and ride its coattails, processing mRNA while some of it is still being transcribed The mRNA can only exit the nucleus whennuclear pore proteins detect the methylated guanosine cap, cap-binding proteins, poly-A tail, and exon junction complex proteins □ Cap-binding proteins later are replaced by translation initiation proteins Regulating Gene Transcription: • In this section, there are lots of similar terms for DNA regions and proteins with special functions; I'll color-code accordingly to help clarify which are which • Enhancer regions (located upstream/before the TATA box and gene to transcribe) bind activator proteins, which help the mediator protein set up the RNA polymerase complex ○ Activator proteins bind specifically to the DNA sequence of their enhancer regions • Repressor proteins can get in the way of RNA polymerase assembly and transcription ○ Repressor proteins bind to the silencer region ○ Note that neither enhancers nor repressors bind to the promoter region; transcription factors and RNA polymerase do • Transcription is regulated by a complex "team," with various specialized roles that help in different ways (and some that instead slow progress, just like teams in real life) ○ Transcription factors, histone modifiers, and the chromatin remodeling complex can all bind to DNA sequences or to the mediator ○ Regulation with many variables or inputs is called combinatorial control and allows fine-tuning the rate of transcription • Some regulatory proteins allow the expression of genes that make other regulatory proteins that allow the expression of genes that do all kinds of other stuff and make more regulatory proteins that…and so forth ○ These are sometimes called linchpin/keystone transcription factors ○ In Drosophila (fruit flies), the protein ey is normally expressed in the eye region but can make a replica eye on the fly's leg if expressed in the leg ○ MyoD causes a chicken fibroblast to become a muscle cell line, which is useful for growing flavorless chicken meat in the lab Translation of mRNA to Protein: • Every group of three nucleotides is called a codon and corresponds to one amino acid ○ The "reading frame" is the three-nucleotide segment that a ribosome "sees": this can start anywhere (123 456 789, 234 567 890, etc.) if not regulated ○ There are 64 codons (combinations of three total nucleotides, 4 ) and only 20 amino acids: the genetic code is redundant 3 of the 64 codons are stop codons Amino acids are carried to the ribosome by tRNA, which is 70-90 nucleotides long and literally kind of t-shaped ○ Amino acids are carried to the ribosome by tRNA, which is 70-90 nucleotides long and literally kind of t-shaped The bottom of the t has an anticodon, a 3-nucleotide sequence complementary to the codon The top of the t (at the 3' end) carries an amino acid encoded by the corresponding codon □ tRNA is "charged" by an aminoacyl-tRNA synthetase, which has binding sites for the amino acid and the anticodon on the right tRNA ATP is hydrolyzed to make the amino acid bond to the tRNA The tRNA synthetase enzyme has an editing site that will remove an incorrect amino acid ◊ A problem with this enzyme causes "sticky mutation" and neurodegeneration in mice The codon selectively binds to the complementary anticodon, which puts the tRNA's amino acid right next to the one before it □ Wobble: some tRNAs can bind codons with a mismatched third nucleotide • Ribosomes are made of many proteins and a few rRNA molecules (weighing 4200 kDa) ○ The large subunit catalyzes peptide bond formation 49 proteins, 3 rRNA molecules, weighs 2800 kDa ○ The small subunit matches tRNA to codons 33 proteins, 1 rRNA molecule, weighs 1400 kDa Has a binding site for mRNA ○ Methionyl-tRNA binds to initiation factors and the small subunit first Then mRNA binds to the small subunit Methionyl-tRNA and the small subunit move along mRNA until the tRNA's anticodon base-pairs with a start codon (AUG) Initiation factors leave, and the large subunit binds to form a complete ribosome ○ The ribosome has three tRNA binding sites, mostly in the large subunit Aminoacyl-tRNA binding site binds a tRNA carrying its amino acid, straight from the cytoplasm Peptidyl-tRNA binding site binds a tRNA attached to the amino acid that just attached to the polypeptide □ The formation of the new peptide bond is catalyzed by a ribozyme--rRNA with catalytic function Exit site binds the tRNA whose amino acid just dissociated from it to bind to the next amino acid □ Image property of Garland Science ○ Termination: instead of an aminoacyl-tRNA, a release factor is complementary to the stop codon (UAA, UGA, or UAG) in the A site and causes hydrolysis of the peptidyl-tRNA bond in the P site to release the polypeptide ○ Just like with transcription of a gene, multiple ribosomes can translate a single mRNA at once (polyribosomes) ○ The protein begins folding while it's still being synthesized Proteins called chaperones (like HSP70) help make sure the protein folds correctly Proteins called chaperones (like HSP70) help make sure the protein folds correctly ○ Know that the "beginning" of a polypeptide chain is the N-terminal (for the nitrogen in the amino group), and the "end" is the C-terminal (for the carbon in the carboxylic acid group) Polypeptides are read N to C like mRNA is read 5' to 3' Protein Degradation and Turnover: • Proteins that folded wrong or are only needed for short periods of time are tagged with ubiquitin (a marker protein) and broken down by the proteasome, a big protein complex with a central cylinder (20S core) containing a protease, between two 19S caps that recognize the ubiquitin • Proteases hydrolyze peptide bonds to generate amino acids that can be reused • This is one of many ways to control the amount of a protein present in a cell Technique: CRISPR The explanation in the lecture notes is…fun. Rather than try to recap that, I highly recommend this article: http://gizmodo.com/everything-you-need-to-know-about-crispr-the-new-tool-1702114381 The video embedded is also nice--here's a direct link to start at the beginning: https://www.youtube.com/watch?v= 2pp17E4E-O8 As always, thanks for reading and happy studying! Don't forget to watch for my Exam 1 Study Guide, with helpful charts made for cramming and detailed explanations of review problems.
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