Brandl Course Summary

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Biochemistry 2280A
Christopher Brandl

Brandl Course Notes Summary Topic 22 - Overview  Humans – 25k genes, ~5-10k are expressed in any tissue  Cancer is caused by defects in tumor suppressors and oncogenes (transcription factors)  Central Dogma: DNA =transcription> RNA =translation> Protein  RNA intermediary allows amplification and can be rapidly degraded which allows genes to be shut off. Gene Regulation in a Eucaryotic Cell 1. Rate of Transcription (broken down into initiation, elongation, termination) 2. Rate of RNA processing (converting a RNA into a translatable molecule, includes 5’ capping and 3’ Polyadenylation, splicing of introns, editing) 3. Rate of transport of mRNA out of nucleus 4. Rate of mRNA degradation 5. Rate of Translation (initiation, elongation, termination) 6. Protein processing (cleavage, phosphorylation, glycosylation, acetylation) 7. Protein degradation Topic 23 – Transcription Promoter – DNA sequence required for transcriptional initiation of a gene. Includes sequences that recognize RNA polymerase and recognize any gene specific regulatory factors. Terminator – DNA sequence required for transcriptional termination RNA Polymerase – Enzyme that catalyzes transcription Regulatory Proteins – Transcription factors, key molecules in differential transcription of genes Bacterial Transcription  RNA Polymerase o Large enzyme with 4 subunits (2 aplha, 1 beta and beta’) o Lacks promoter specificity (cannot recognize promoter sequences) o Holoenzyme transcribes RNA specifically from promoters o Beta and beta’ subunits contain a sigma subunit that recognizes promoter structures and decreases binding to non-promoter DNA  Initiation of Transcription 1. RNA polymerase recognizes and binds to promoter (closed complex) via the sigma factor (base specific contact) 2. Polymerase unwinds the DNA at the transcriptional start site (open complex) 3. NTP is brought to the template, no primer required 4. Chain elongation begins and proceeds 5’-3’ 5. After about 5-10 nucleotides sigma falls off 6. The transcription bubble moves along with the template DNA reannealing behind 7. Chain elongation continues until a terminator, polymerase falls off  Structure of Bacterial Promoters o Recognition sites on bacterial DNA recruit RNA polymerase holoenzyme o Found at approximately -35 (TTGACA) and -10 (TATATT), 5’-3’  These are consensus statements, ie. the most common base pair for each position of a list of promoters  Regulation of Transcription 1. Strength of basic promoter elements 2. Gene specific regulatory proteins  Trp Operon o Genes required for tryptophan biosynthesis in E.coli are transcribed as a single unit from a common promoter. 5 proteins are translated. This is called an operon and is only found in bacteria. o When tryptophan concentration is high the Trp repressor binds tryptophan. A conformational change allows the repressor to bind to the operator site and inhibits RNA polymerase from transcription. o Trp Repressor has a 107 amino acid residues and a helix turn helix motif required for DNA binding. Binds to a major groove as a dimer.  Positive Control (Lac Operon) o Increased transcription is accomplished by increasing rate of recruitment of RNA polymerase o Usually achieved through protein-protein interactions between activator and enzyme o Lac Operon  Contains genes required for metabolism of lactose  Negative regulation similar to the Trp operon  In the absence of lactose the Lac repressor binds the promoter (-10 sequence), inhibiting action of RNA polymerase  When lactose is present it binds to the Lac repressor. Conformational changes occur and the protein no longer binds to the operator, allowing the sequence to be transcribed.  Glucose control also occurs o In the presence of glucose genes required for metabolism of Lac are shut off (catabolite repression) o When glucose is low, cAMP rises. When cAMP is high CAP binds to it. CAP with cAMP binds to the -35 sequence of the Lac operon and stimulates recruitment of RNA polymerase to the promoter. Differences in Transcriptional Regulation between Procaryotes and Eucaryotes 1. Eucaryotes have three different RNA polymerase  Polymerase I – For ribosomal RNA (rRNA)  Polymerase II – For messenger RNA (mRNA)  Polymerase III – For tRNA and snRNA 2. Do not have operons (genes are transcribed as single units, monocistronic) 3. Promoter recognition is through a distinct set of proteins  TATA-binding protein binds to the TATA-box, ~20 basepairs upstream from the transcriptional start site 4. Regulatory elements located thousands of base pairs away  DNA looping 5. Nucleosomes and Chromatin Structures affect access of transcriptional factors to DNA  Post-translational modification of histones affects structure 6. Combinational Control  Groups of proteins work together to determine expression of one gene  Other DNA binding motifs o Zn finger, Homeodomain, Leucine zipper, Helix loop helix (all function as dimers and generally make contact with the major groove)  Regulation of Genes for Galactose Metabolism in Yeast o GAL1, GAL2, GAL10, GAL7 are controlled both positively and negatively. Each is encoded by its own mRNA. o Positive Regulation of GAL Genes  Gal4 (protein) binds as a dimer to a 17bp sequence. GAL10 has four of these recognition sites (UASGAL).  In the absence of galactose Gal4 is bound to Gal80, which blocks the activation function.  When galactose is present Gal3 binds galactose and interacts with Gal80 to free Gal4 o Negative Regulation of GAL Genes  Occurs whether or not galactose is present  In the presence of glucose Mig1 binds the GAL10 promoter and inhibits transcription.  When glucose is not present Mig1 becomes phosphorylated and cannot enter the nucleus. Topic 24 – RNA Processing in Eucaryotes 1. Capping of the 5’ End  7 methyl guanosine  Required for export from the nucleus, aids in stabilizing mRNA from being degraded, translational signal 2. Polyadeylation  Addition of a long A tail to the 3’ end of RNA  RNA is cleaved after an AAUAAA sequence and 300 As are added  Provides additional stability by reducing effects of 3’ exonuclease, as a role in nuclear export and translation 3. Splicing  Coding sequences are interrupted by one or more non-coding regions called introns (exons = coding)  Provide opportunity for differential splicing, ie. the mRNA can be put together in different ways Removal of Introns  The Spliceosome o snRNPs are complexes of RNA and protein o RNA component has recognition function and base pairs to precursor mRNA o Splicing occurs by 2 transesterifictation reactions  Cleavage at the 1-intron boundary from attack of 5’ splice junction by adenine in the branch point (generates a lariat structure)  3’-OH of exon 1 reacts with the 3’ splice junction, cutting out the intron and joining the exons  The lariat is displaced and degraded  Self-Splicing Introns o In pre mRNA the snRNPS are not required. RNA is capable of catalyzing its own conversion to mRNA o This is an example of RNA having catalytic potential (another is ribozymes, which catalyze cleavage of other RNA molecules in sequence specific fashion) RNA Export  In eucaryotic cells RNA is synthesized in the nucleus and translated in the cytoplasm  Nuclear pores transport mRNA  Regulated proecss that requires recognition of proteins bound to the poly A-tail and the 5’ cap, as well as internally Topic 25 – Translation Genetic Code – mRNA spells out amino acid code in 3 letter codons. Each protein hasa a specific reading frame.  Universal  Non-overlapping  Commaless (no gaps)  61 codons for 20 amino acids, ie. redundant  3 stop codons, 1 start codon  More common amino acids have more codons  Related acids have similar codons tRNA  Translation requires tRNA molecules, which are vehicles that bring amino acids to the growing peptide chain  Codon specific fashion relying on base pairing  About 80 nucleotides in length and have a cloverleaf secondary structure  Anti-codon of tRNA hybridizes with the codon  The correct amino acid is covalently linked to the 3’ end of the tRNA  Wobble occurs because accurate base pairing for many tRNAs only requires matching at the first two posititons Amino Acid Activation – Aminoacyl tRNA Synthetases  ATP provides energy for carboxyl of an amino acid to couple to the 3’ end of a specific tRNA 1. Provides an energy source for later peptide bond formation 2. Provides specificity by matching the correct amino acid to the specific tRNA Ribosomes  Large protein-RNA complexes  Large (49 proteins, 3 rRNA) and small (33 proteins, 1 rRNA) subunits o Small subunit matches tRNA to the codon o Large subunit catalyzes formation of peptide bonds  Three sites for tRNA o A site (aminoacyl tRNA site) o P site (peptidyl tRNA site) o E site (exit site) Process of Translation  A tRNA in the P site is linked to the growing polypeptide  An aminoacyl tRNA accesses the A site per base pairing rules  Energy of the aminoacyl-tRNA in the P site is used fo form a peptide bond between the amino group of the amino acid in the A site with the carboxyl group of the amino acid in the P site  The reaction is coupled with a conformational change in the ribosome that shifts the small subunit and the cycle continues Initiation of Translation  Begins at an AUG with a special tRNA that carries Methionine  Small subunit recognizes the 5’ cap of mRNA and moves in a 5’ to 3’ direction until it reaches an AUG codon  Initiation factors then dissociate allowing the large subunit to bind the small subunit. In this process the initiator tRNA is positioned at the P site  In bacteria multiple reading frames are found in a single message. This means more than one protein must be translated from RNA. Ribosomes can initiate translation at internal AUGs Termination of Translation  Stop codons signal end of translation  Release factors associate with ribosome when any stop codon reaches the A site. Peptidyl transferase than adds a water to the end of the chain. Antibiotics and Translation  The majority of known antibiotics block translation  Exceptions include penicillin, ampicillin Topic 26 – Recombinant DNA Technology and Genetic Engineering  Techniques by which DNA fragments from different sources are recombined to make a new DNA molecule.  Selective breeding are examples of manupulating genes, but this is a slow process.  In research, o Allowed the expansion of scientific knowledge in areas of gene structure and function o Power of RDT reflects link between genes and proteins o Site directed mutagenesis, protein overexpression, protein engineering o Complete genome sequences of hundreds of organisms are known  In biotechnology, o Enabled the production of large amounts of rare proteins o Introduced genes across species barriers o Allowed creation of novel proteins, modified organisms and even novem organisms o Medicine – Drug production and creation, diagnosis, genetic counseling and screening, gene therapy o Agricult
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