BIO230 Midterm review notes with highlights

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University of Toronto St. George
Darrel Desveaux

Tuesdays RW429 Tuesday 2-4 pm Dr. Garside Regulation of Genome expression Ch 1. Pg. 1-8, 14-16 Tree of life: Eubacteria, Archaebacteria, Eukaryotes At a molecular level, archaea seem to resemble eukaryotes more closely in their machinery for handling genetic information (replication, transcription, and translation), but bacteria more closely in their apparatus for metabolism and energy conversion. 1) Prokaryotic cells: Eubacteria, and archaea Single celled Lack nucleus and organelles -Plasma membrane (selectively permeable), DNA localized in nucleoid but no membrane surrounding it, cell wall (sugars), appendages (e.g. flagellum) 2) Eukaryotic cells Plants, fungi, animals, humans Single-celled (e.g. yeast) or multicellular (e.g. humans) Have nuclei and organelles -About 1000x bigger in volume than prokaryotic cell -Possibly evolved through phagocytosis of other prokaryotic cells All known life forms possess a genome (e.g. human genome ~25,000 genes; at any one time only 30-60% of genes are expressed) Genome: encodes the information to construct and maintain an organism Most genomes are made of DNA (some viruses have RNA genomes) Release of the biological information stored in the genome requires genome expression The first product of gene expression: the transcriptome (maintained by the process of transcription) The repertoire of RNA molecules present in a cell at a particular time Visualized by DNA microarray (differences in abundance; red = high, green = low) The second product of genome expression: the proteome (maintained by the process of translation) The collection of proteins in a cell; define the biochemical functions of the cell Visualized through: e.g. 2D gel electrophoresis Genome (DNA)…Transcriptome (RNA)…Proteome (proteins) = central dogma Different cell types of a multicellular organism contain the same genome. How to produce different cell types? Differences in genome expression. Expression of almost all genes varies from one cell type to another. Genome expression is regulated at many steps from DNA to protein. Proteome…Interactome…Metabolome Prokaryotic transcriptional regulation Ch.7 pg. 411-416, 432-438 Regulation of gene expression is crucial for: -Responses to extracellular stimuli (both multicellular and unicellular organisms) -Defining cell types (multicellular organisms) -Also in defining and identifying disease cells e.g. cancer DNA is transcribed into RNA by the enzyme RNA polymerase -Energy in phosphate bonds -Prokaryotic transcription requires sigma factor -Promoter region: region of DNA that indicates transcription start site -RNA polymerase holoenzyme -Transcription begins, rather inefficient -Once ~10 nucleotides are synthesized, sigma factor is released -Transcription elongation, more efficient -Transcription termination at DNA termination signal How are genes transcribed at different efficiencies? Gene expression in prokaryotes and eukaryotes is regulated by gene regulatory proteins (transcription factors), which bind specifically to regulatory regions of DNA (cis elements) Gene regulatory proteins can turn genes ON (positive regulator): activators OFF (negative regulator): repressors (e.g. Trp operon) Gene regulatory proteins discovered using bacterial genetics E. Coli: -Unicellular prokaryote -One chromosome of circular DNA -Encodes about 4300 proteins -Many are transcriptionally regulated by food availability Prokaryotic feature: -Multiple genes can be transcribed into a single RNA molecule called an operon -Rare in eukaryotes -Transcribed by one promoter E.g. Tryptophan operon -5 Genes -Encode enzymes for tryptophan biosynthesis -Transcription regulated by a single promoter -Gene activated when there is not enough tryptophan in the environment -Two protein-bound states: 1) Bound by RNA polymerase (Trp gene expression ON) 2) Operator bound by tryptophan repressor protein (Trp gene expression OFF) Trp repressor binding blocks promoter access Negatively regulates Trp expression BUT Trp repressor DNA binding activity is regulated Must bind two molecules of trp to bind DNA Repressor and operator provide a simple switch to control Trp biosynthesis Trp repressor (dimer) contains a helix-turn-helix -Binds in major groove of DNA double helix -Tryptophan binding induces conformational change so that it fits in major groove E.g. Lac operon -3 genes required for transport of lactose into the cell -Enables use of lactose in the absence of glucose -Dual regulation: both positive and negative control 1) Activator: Catabolite Activator Protein (CAP) promotes Lac expression (low glucose, high lactose) -CAP binding site is upstream of RNA polymerase binding site and operator 2) Repressor (tetramer): Lac Repressor Protein inhibits Lac expression (low lactose) -When lactose levels are low, Lac repressor is bound to the operator; Lac operon gene expression is OFF (but never completely) -Increases in lactose increase levels of allolactose, related to lactose; -Requires B-galactosidase (1 gene of Lac operon encodes B-galactosidase, breaks down lactose to glucose and galactose) -Allolactose binds to Lac repressor: conformational change, decreases DNA-binding activity, releases Lac repressor from the operator Why need an activator? -RNA polymerase binding to Lac promoter is inefficient -Efficient RNA polymerase binding to Lac promoter requires CAP to be bound -CAP contains a helix-turn-helix DNA binding domain -CAP DNA-binding activity is activated by low glucose -Decreasing glucose levels increase levels of signaling molecule called cyclic AMP (cAMP) -cAMP binds CAP protein: conformational change, increases DNA-binding activity, binds to CAP-binding site -CAP recruits RNA polymerase to the Lac promoter -CAP makes it favourable for RNA polymerase to bind Under conditions where both glucose and lactose levels are low you would expect expression of the Lac operon to be: OFF because although CAP is bound, lac repressor is bound to promoter, so polymerase cannot bind Why is it important that expression of the Lac operon is always slightly on? E. Coli needs B-galactosidase encoded by the Lac operon to respond to lactose. SUMMARY: -Negative regulation: competition between RNA polymerase and repressor protein for promoter binding -Positive regulation: activator protein recruits RNA polymerase to activate transcription Regulatory elements can also be found: far upstream of gene (prokaryotes and eukaryotes), downstream of gene (eukaryotes) and within gene (introns; eukaryotes) Some regulatory elements are distant from the transcriptional start site and influence transcription (i.e. Lac operon): -DNA looping -Lac repressor is a tetramer and can bind two operators simultaneously -Increases affinity of Lac repressor for the Lac promoter -May increase effective concentration of repressor Bacteriophage Lambda: -Virus that infects bacterial cells -Positive and negative regulatory mechanisms work together to regulate the lifestyles of bacteriophage lambda -Two proteins repress each other’s synthesis Bacteriophage lambda can exist as one of two states in bacteria: -Favourable conditions: integration of lambda DNA into host chromosome (prophage pathway) -Damaged/unfavorable conditions: synthesis of viral proteins needed for formation of new viruses, rapid replication and packaged, new bacteriophage released by cell lysis (lytic pathway) Lambda repressor (negative) -Made in prophage state -Occupies the operator -Inhibit expression of Cro, positively regulates its own expression -Most bacteriophage DNA not transcribed Cro protein (activator) -On in lytic stage -Occupies the operator (different location than lambda repressor) -Blocks synthesis of repressor protein, allows for its own synthesis -Does not actively recruit RNA polymerase, but by blocking the repressor it allows its own synthesis -Most bacteriophage DNA is extensively transcribed What triggers switch between states? -Host response to DNA damage (e.g. damage control proteins) inactivates repressor (switch to lytic state) -Under good growth conditions, repressor protein turns off Cro and activates itself (positive feedback loop), amplification effect, maintains prophage state Transcriptional circuits: positive/negative, flip-flop device, feed-forward loop -Positive feedback loop can be used to create cell memory -Feed-forward loops can measure the duration of a signal (-Brief input: B does not accumulate, so no transcription of Z; -Prolonged input: B accumulates, A+B activate Z; -Eliminate noise in environment) Combinations of regulatory circuit in eukaryotic cells: exceedingly complex (e.g. sea urchin embryo development) Scientists can construct artificial circuits and examine their behavior in cells (form of biological engineering: synthetic biology) Synthetic biology: E.g. create a simple gene oscillator using a delayed negative feedback circuit “the repressillator” 1) A expressed, represses B 2) B repressed, so C is not repressed 3) C expressed, represses A (and so on and so forth) Oscillation In bacteria: does oscillate, increasing amplitude due to bacterial growth Natural example: feedback loops carry out 24-hour cycle in Drosophila: delayed negative feedback loop (per gene, tim gene) Clicker question: Flip flop device not shown Transcription Attenuation -Premature termination of transcription -RNA adopts a structure that interferes with RNA polymerase -Regulatory proteins can bind to RNA and interfere with attenuation -Prokaryotes, plants and some fungi also use riboswitches (small molecule) to regulate gene expression Riboswitches: -Short RNA sequences that change conformation when bound by a small molecule -E.g. Prokaryotic riboswitch that regulates purine (A, G) biosynthesis: -Low guanine levels: transcription of purine biosynthetic genes is ON -High guanine levels: guanine binds riboswitch -Riboswitch undergoes conformational change, causes RNA polymerase to terminate transcription; transcription of purine biosynthetic genes is OFF Eukaryotic gene regulation Cells produce several types of RNA (E.g. mRNA, rRNA, tRNA) Types of RNA polymerase in eukaryotes: RNA polymerase I: rRNA RNA polymerase III: tRNA RNA polymerase II: all protein coding genes -Requires 5 general transcription factors: TFIID, TFIIB, TFIIF, TFIIE, TFIIH -Prokaryotes only need one; sigma factor Eukaryotic -Genomes lack operons -DNA packed into chromatin, provides additional mode of regulation -Transcriptional activation requires many gene regulatory proteins Transcription initiation in eukaryotes requires many proteins: -General transcription factors (E.g. TFIID, TFIIH) -Help position RNA polymerase at eukaryotic promoters (contain TATA box) -Required by nearly all promoters used by RNA polymerase II Mediator acts as an intermediate (doesn’t bind directly to DNA) between regulatory proteins and RNA polymerase Eukaryotic gene regulatory proteins (~2000 in human genome) -Often function as protein complexes -Can act over very large distances, sometimes >10,000 bp away -DNA looping is one mechanism Coactivators and corepressors assemble on DNA-bound gene regulatory proteins, but do not directly bind DNA Eukaryotic Activator Proteins -Modular design: 1) DNA binding domain (DB) recognizes specific DNA sequence 2) Activation domain (AD) accelerates rate of transcription 1 and 2 can mix and match -Attract, position and modify: 1) General transcription factors 2) Mediator 3) RNA polymerase II Directly (acting on above components, attract them to promoters) or indirectly (modifying chromatin structure) Nucleosomes: basic structure of eukaryotic chromatin -DNA wound around a histone octamer (two sets of H2A, H2B, H3, H4) -“Beads on a string” with linker DNA -Activator proteins can alter chromatin structure and increase promoter accessibility, because transcriptional machinery cannot assemble on promoters tightly packaged in chromatin -Nucleosome structure can be altered by chromatin remodeling complexes in an ATP-dependent manner: 1) Nucleosome sliding, 2) Nucleosome removal 3) Histone exchange (2 & 3 require cooperation with histone chaperones) Four major ways activator proteins alter chromatin: Chromatin-remodeling complex: 1. Remodeled nucleosomes 2. Histone removal 3. Histone replacement 4. Histone-modifying enzyme (signal for chromatin remodeling): -Histone code: specific modifications to histone tails by histone modifying enzymes called “writers” -Code “reader” proteins recognize these modifications; provide meaning to the code (different modifications mean different things) -Phosphorylation (addition of phosphate group) by enzyme kinase -Acetylation (addition of acetyl group) by enzyme acetyltransferase -Methylation (addition of methyl group) by enzyme methyltransferase (methylase) Histone modifications occur on specific amino acids of histone tails E.g. human interferon gene promoter -Transcriptional regulation using the histone code 1. Activator protein binds to chromatin DNA and attracts a histone acetyltransferase (HAT) 2. HAT acetylates specific lysine 9 of H3, lysine 8 of H4 3. Activator protein attracts a histone kinase (HK) 4. HK phosphorylates serine 10 of H3, can only occur after acetylation step 5. Phosphorylation modification signals HAT to acetylate lysine 14 of H3 Histone code for transcription is written 6. TFIID and a chromatin-remodeling complex bind to acetylated histone tails, initiate transcription Eukaryotic transcriptional repression UNLIKE prokaryotes, eukaryotic repressor proteins rarely compete with RNA polymerase for access to DNA -Instead, use a variety of mechanisms to inhibit transcription: 1. Interfering with activator function (compete with activator for DNA binding) 2. Masking the activation surface 3. Direct interaction with general transcription factors 4. Recruitment of chromatin remodeling complexes 5. Recruitment of histone deacetylases 6. Recruitment of histone methyltransferases Histone “reader” and “writer” proteins can establish a repressive form of chromatin (guided by gene regulatory proteins) -Spreading the histone code along chromatin carried out by “reader-writer” complexes -DNA methylase is attracted by “reader”, and methylates nearby cytosines in DNA -DNA methyl-binding proteins bind methyl groups; stabilize structure -Methylation and gene expression patterns can be inherited: epigenetic inheritance Post-Transcriptional Regulation Transcriptome analysis (e.g. microarray) provides a “signature” of cell type and cell state: -Response to extracellular stimuli -Disease states e.g. cancer Experiment: Primary acute myelogenous leukemia cells: transform from undifferentiated cells to differentiated neutrophils using chemicals IC50: concentration of compound required to inhibit cell proliferation by 50% After application of chemicals, can inhibit proliferation Prokaryotes: simple transcription to translation Eukaryotes: pre-mRNA 1) Covalent modifications of RNA ends 2) Removal of intron sequences RNA capping: -Addition of a modified guanine nucleotide to the 5’ end of pre-mRNA (3 enzymes involved) -Cap bound by cap-binding complex (CBC) Functions: -Helps in RNA processing and export from the nucleus -Important role in translation of mRNAs in the cytosol -Protects mRNA from degradation RNA splicing: Eukaryotic genes: exons (expressed) and introns (non-coding, to be spliced out) -Some exons are non-coding but are not spliced out Both are transcribed into RNA but the introns are removed in a process called RNA splicing -Different cells can splice an RNA transcript differently to make different proteins from the same gene: alternative splicing (isoforms) ~75% of human genes produce multiple proteins Therefore size of proteome is not necessarily the size of the genome Carried out by an enzyme complex made up of RNA and proteins termed the spliceosome -Sites of proper splicing are bound (after splicing) by exon junction complexes (EJC): serves as marker for properly spliced RNA Alternative splicing can be regulated a) Negative control, repressor: no splicing b) Positive control, activator: splicing Usually with a combination of negative and positive E.g. drosophila sex determination: -Ratio of X chromosome to autosomal sets X:A = 0.5 (male, default) X:A = 1.0 (female) Three genes involved, regulated by alternative splicing: Sex-lethal: splicing repressor (negatively regulated) Transformer: splicing activator (negatively regulated) (Both regulate): Doublesex: regulates sex gene expression All 3 genes contain regulated splice sites Male: (default pathway because no regulation) X:A=0.5 Sex-lethal: No regulation of splicing, splice product is non-functional Transformer: no regulation of splicing, splice product is non-functional Double sex: no regulation, activated by transformer, represses female gene expression Female: (regulated splicing) X:A=1.0 1 step: special sex lethal (repressor) produced (alternative internal promoter site) that is functional when spliced (transient), regulates itself -Functional Sxl protein represses splicing of Sxl and Tra -Functional Tra protein activates splicing of Dsx -Positive regulator of splicing: Dsx protein represses male gene expression 3’ Polyadenylation -More complex than transcription termination in prokaryotes -Signals encoded in genome: -RNA polymerase transfers protein complexes to RNA: CstF (cleavage stimulating factor) and CPSF (cleavage and polyadenylation specificity factor) -RNA cleaved -Transcription terminates -Poly-A polymerase (PAP) adds ~200 A nucleotides (from ATP) to the 3’ end of RNA (not genome-coded) -Poly-A tail is bound by poly-A binding proteins -Aid in: RNA export, translation, mRNA stability Eukaryotic RNA processing is initiated before transcription is complete Coupling transcription and RNA processing: -During transcription elongation the C-terminal domain (CTD) of RNA polymerase binds RNA processing proteins and transfers to RNA at appropriate time -Binding of RNA processing proteins is regulated by phosphorylation of RNA polymerase (patterns of phosphorylation signal for different proteins) RNA transport from the nucleus: -The cell selectively transports mature mRNA from the nucleus Markers of mature mRNA must be acquired for export -Cap binding complex (CPC) -Exon junction complexes (EJC) -Poly-A binding proteins These proteins travel with the mRNA to the cytosol Markers of immature mRNA must be lost for export: -Proteins involved in RNA splicing (e.g. snRNPs) Only ~1 out of 20 RNA leaves the nucleus, makes it difficult for pathogens to hijack Improperly processed mRNAs will eventually be degraded in the nucleus by the exosome E.g. human AIDS virus HIV: retrovirus (reverse transcriptase creates a double stranded DNA molecule from an RNA molecule) -HIV transcript is spliced in different ways to produce over 30 different mRNAs: some retain introns and will not be exported to cytosol for translation -Rev protein in HIV: mRNA has no introns -Binds Rev responsive element (RRE) in unspliced RNAs -Interacts with nuclear export receptor -Directs export of unspliced mRNAs -Late HIV synthesis mRNA quality control: (tightly coupled to translation) -Some mRNAs are incompletely processed or damaged in the cytosol -Need to prevent production of aberrant protein that can be toxic to cells -tRNAs match amino acids to codons (3 nucleotides) in the mRNA: genetic code -mRNA message is decoded in ribosomes made up of >50 different proteins and several RNA molecules (A, P, E sites) -Amino acids are added to the C-terminal end of the growing polypeptide chain -Therefore proteins are synthesized from N-to C-terminus Eukaryotes: -Translation initiation machinery recognizes 5’ cap and poly-A tail -Eukaryotic initiation factors -5’ cap bound by eIF4E, displaces CBC -Poly-A binding protein bound by EIF4G -Recruit small ribosomal complex, which will initiate translation at first AUG downstream of 5’ cap (some exceptions) -Ensures that both ends of mRNA are intact -Exon junction complex (EJC) also stimulates translation ensuring proper splicing Nonsense-mediated mRNA decay (eukaryotes) -Prominent mRNA surveillance system -Surveys for nonsense (STOP) codons in the “wrong place” -Indicator of improper splicing (accidentally shifted frame) -May have played an important role in evolution of eukaryotes by allowing selection of DNA rearrangements or alternative splicing patterns that produce full-length proteins -Important role in cells of the immune systems where extensive DNA rearrangements occur to produce antibodies -Plays a role in many human diseases: caused by mutations that produce aberrant proteins; cells can degrade aberrant mRNA and allow functional protein to accumulate Normal splicing: -Ribosome binds mRNA as it emerges from nuclear pore -EJC are displaced by moving ribosome -Stop codon is in last exon -No EJC bound when ribosome reaches stop codon -mRNA released in cytosol Abnormal splicing: -Ribosome binds mRNA as it emerges from nuclear pore -EJC are displaced by moving ribosome -Stop codon is premature -EJC still remain on mRNA when ribosome reaches stop -mRNA degraded (mediated by Upf proteins) Prokaryotes: -Also has quality control for incomplete or broken mRNA -Ribosome stalls on broken or incomplete mRNAs and do not release -A special RNA tmRNA (carries an alanine amino acid) is recruited to A site -Broken mRNA is released -Alanine added onto polypeptide from tmRNA; acts like a tRNA but with no codon -Ribosome translates 10 codons from tmRNA; acts as an mRNA -11 amino acid tag (ala +10 codons) is recognized by proteases that degrade the entire protein mRNA stability: -In prokaryotes, exonucleases rapidly degrade most mRNAs (from the ends) In eukaryotes, mRNAs are more stable and degradation is regulated: -Two main mechanisms: both involve gradual poly-A tail shortening -Exonuclease (deadenylase) when mRNA reaches cytoplasm (acts as a timer for mRNA lifetime) -Tail shortened from 200 to ~25 A in humans, degradation: decapping and 5’ to 3’ degradation, or rapid 3’ to 5’ (both can occur on the same mRNA) -Cytoplasmic poly-A elongation can also occur to stabilize mRNA -Proteins also interfere with poly-A shortening E.g. Transferrin receptor imports iron into cell (needed when cellular iron is low) -Addition of iron into cell decreases transferrin mRNA stability, less receptor made -Transferrin mRNA stabilized by cytosolic aconitase (binds 3’ UTR, increase receptor production by blocking endonucleolytic cleavage site) -When there is excess iron: aconitase binds iron, conformational change decreases affinity for transferrin receptor, mRNA released -Exposes 3’ UTR endonucleolytic cleavage site: poly-A removed, mRNA degraded Competition between mRNA translation and mRNA degradation: -Deadenylase that shortens poly-A tail binds 5’ cap (like eIF4E) miRNAs -Non-coding RNAs called micro RNA (miRNA) also regulate mRNA stability (~400 in humans) -miRNAs base-pair with specific mRNAs -Precursor miRNA synthesized by RNA polymerase II (forms a double strand with itself in the nucleus), and undergoes 5’ cap and poly-A -After special processing (“cropping”, “dicing” by dicer enzyme), miRNA associates with a protein complex called RNA-induced silencing complex (RISC) -A protein of RISC called Argonaute cleaves and discards one strand of miRNA -RISC seeks for mRNA with complementary nucleotide sequences -Argonaute plays a critical role in base-pairing miRNA with mRNA -Two possible outcomes: extensive match (degradation), or less extensive match (reduced translation, transfer of mRNA into P-bodies, eventual degradation) -A single mRNA can regulate a whole set of different mRNAs with a common UTR sequence -Proteins used in miRNA regulatory mechanisms also serve as a defense mechanism against foreign RNA molecules -RNA interference (RNAi) -Found in fungi, plants -Many viruses and transposable elements produce double-stranded RNA as a part of their life cycles -RNAi destroys double-stranded RNA, initiated by Dicer protein complex into siRNA -siRNAs (small interfering RNAs) bound by Argonaute and RISC proteins that cleave it to single stranded, and follow miRNA route to destroy complementary RNA molecules produced by the virus siRNAs can also regulate transcription: -siRNAs interacts with Argonaute and RNA induced
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