Genetics Exam 2 PDF

General success tips: This test has so much information, and the key is to organize that information. I believe the best way to organize it is as follows: Genes - Gene structure - Prok - Euk - Similarities and differences between Prok and Euk - Gene expressi...

General success tips: This test has so much information, and the key is to organize that information. I believe the best way to organize it is as follows: Genes - Gene structure - Prok - Euk - Similarities and differences between Prok and Euk - Gene expression (synonymous with translation and transcription) - Prok - Euk - Similarities and differences between Prok and Euk - Expression regulation: Prok vs Euk - Prok - Operons - Euk - Similarities and differences between Prok and Euk Mutations - Pure memorization of vocab terms - She will test your knowledge of other things by asking how a loss of function mutation effects certain things, see my practice test Molecular cloning - Conceptual (process including knowing enzymes and what colors indicate) - Practical (being able to choose proper enzyme and give final expected band sizes) Not all topics are made equal. Here is the list of essential topics you MUST know: - General gene parts (promoter, transcription start site, coding region, etc.) in both euk and prok. - Transcription initiation, elongation, termination for prok, and transcription initiation and termination for euk - Translation initiation, elongation, termination for prok - All types of mutations - Conceptual and practical parts of molecular cloning - Lac operon, trp operon in very high detail, and general CAP operon info Here are the topics you need to know less about (general idea, not as in depth): - mRNA processing - Alternative splicing - Translation for euk - Loss/gain of function in mutations - Euk gene expression - Chromatin conformation - Epigenetic profile and imprinting - X-inactivation  - Transcription regulation in initiation - Waddington’s epigenetic landscape - Post-transcriptional regulation - Regulation of translation - Prok gene expression - Genome/chromatin structure - Epigenetic profile - Transcription regulation - Post-transcriptional regulation - Translation regulation - Regulation via ncRNAs - Differences in prok and euk gene structure, gene expression (transcription/translation), gene regulation - Be able to name 5 similarities/differences between each topic. Don’t have to explain too much. See directly below for what I memorized for the 3 topics. She WILL make questions that challenge your knowledge between the topics. Don’t double dip, gene expression differences and similarities are mutually exclusive compared to gene regulation stuff, make sure you’ve categorized them properly I would have gotten a letter grade higher had I organized the information like this. 20% of our test was directly on similarities/difference of the previously mentioned gene structure/gene expression/gene regulation. You are entirely capable of succeeding. The first step is organizing what information you plan to tackle, and taking it one topic at a time. Each topic will greatly increase your grade. The first test is not representative of what you’re capable of. Identify what needs work, and put your effort there. If you’re not even sure where to begin, organize. If you feel confident, test your knowledge on the practice test. The best way to prepare after having studied a good deal is to do the practice test or simply write out your understanding. There will be a bit of time crunch, and only after you’ve committed your explanations to paper can you move efficiently and truly have committed the information to memory. If you have any questions, PLEASE reach out, we love to help, and we know you can do it! Random memorization of Prok vs Euk Gene structure compare and contrast Differences 1. Prok: just exons - Euk: introns as well 2. Prok: upstream regulatory sequence - Euk: both upstream and downstream regulatory sequences 3. Prok: poly a tail is not a sequence found on the gene - Euk: has poly a tail sequence found the 3’ UTR 4. Prok: have shine-dalgarno sequence for cotranscription - Euk: has no coupling sequence  Similarities 1. Both have a promoter sequence upstream of coding regions 2. Both have 5’ and 3’ untranslated regions Intron - downstream - poly A - shine - promoter - UTRs I divulge a simple prokaryote region Transcription and translation difference (gene expression) Differences 1. Prok: Only 1 RNAP - Euk: 4 different RNAP with different functions 2. * Prok: produce polycistronic mRNA mostly - Euk: only produce monocistronic mRNA 3. Prok: can initiate transcription with just holoenzyme - Euk: may require mediator proteins 4. * Prok: require either intrinsic or factor dependent termination for transcription - Euk: use cleavage and polyadenylation specificity factor to terminate transcription 5. * Prok: have coupled transcription and translation taking place in the cytoplasm - Euk: have transcription in the nucleus and translation in the cytoplasm 6. * Prok: have smaller ribosome complex (can include 70S) - Euk: have larger ribosome complexes and factors for translation (can include 80S) One - mRNA - holoenzyme - intrinsic - coupled - ribosome One mRNA hangs in cytoplasm readily Regulation of gene expression difference Differences 1. * Prok: does not have mRNA processing - Euk: has many functions of mRNA processing like alternative splicing, 5’ capping, and 3’ polyA tail 2. Prok: DNA is found in nucleoid - Euk: chromatin is divided into unique regulatory complexes in topologically associating domains, altering gene expression rates 3. * Prok: DNA is open to RNAP for recognition and binding - Euk: DNA may be methylated and can pack tight enough to block RNAPs from binding 4. Prok: have a singular promoter sequence - Euk: can have alternative promoter sequences that produce different mRNA, and thus a different protein 5. Prok: has some control of RNAP binding - Euk: have regulatory functions that can prevent RNAP binding and prevent elongation once bound 6. Prok: use regulatory sequences that produce activators and/or repressors that change the amount of expression - Euk: use enhancers and silencers, where silencers may prevent any expression 7. Prok: have a single copy of a chromosome - Euk: have a pair of sex chromosomes where female cells inactivate one chromosome, having difference in expression at the cellular level 8. Prok: have few regulatory proteins - Euk: has complex network of regulatory proteins that can prevent transcription from occurring Processing - TAD - methylation - alternative promoters - binding/elongation - activators/repressors/enhancers/silencers - X inactivation - regulatory complex People differ many aways, because an independent rearrangement complex Strand Names Other names for strands: Coding (5’ to 3’) called plus, sense strand, or non-template. Template strand is minus, antisense strand, and non-coding strand. Called coding strand because it has the same sequence as what is translated from template strand, except uracil instead of thymine Genes - Prokaryote Prokaryotic genes: have promoter, RNA coding region, and transcription terminator Types: Protein coding gene; code for polypeptide, RNA gene; determines order of rNTPs of a functional RNA molecule Transcription Transcription overview - Begins with initiation (promoter recognition, formation of closed complex, formation of open complex). Then elongation. Then termination (either intrinsic or factor dependent) Transcription Initiation - Promoter recognition: Recognized and bound by transcription apparatus. Determines which strand and direction of transcription. Contains cis-acting elements that work together with promoter to regulate gene expression, including UP element, -35 & -10 consensus sequences, Ext, Dis, and CRE. Actual promoter sits in between -35 and -10, and transcription takes place at +1.  - Initiation: RNAP searches for start sequence - Formation of closed complex: Sigmas 3rd domain interacts with EXT element, 4th domain with -35 element of promoter. “Holoenzyme” is combination of sigma subunits and RNAP. Holoenzyme occupies region -50 to +20 of gene to be transcribed - Formation of open complex is last, where sigma’s 2nd domain interacts with non-template strand of -10 element of promoter, driving separation of DNA strands. Once DNA melts to expose template strand, AND 1st DOMAIN of sigma moves out of the way of the active site (that was its purpose), the catalytic site of RNAP becomes “open complex” Video (4:22-8:42): https://www.youtube.com/watch?v=JebsvaBCpQo Transcription elongation  - Holoenzyme produces series of short 2-6 nucleotide transcripts, which are released from template (abortive transcription). Once about 12 ribonucleotides long (true transcription) & RNAP passes promoter, sigma leaves. This is where actual transcription occurs and goes until the terminator. Does not occur at uniform rate. Template sequences and RNA structures can induce pausing or backtracking of RNAP. Video (2:30-end): https://www.youtube.com/watch?v=nXj2Hmd51l4 Transcription Termination - Intrinsic transcription termination: terminator DNA sequence is GC rich palindrome, which causes hairpin loop in RNA transcript. Inside RNAP will be AU rich sequence, weak. Loop destabilizes RNAP complex, utilizes weak AU point, causing mRNA to snap off - Factor-dependent terminator: Rho is a large hexameric protein that physically interacts with growing RNA transcript, does not have a standard DNA sequence it originates from. Rho binds to RNA at “rut” site, and translocates along RNA in 5’ to 3’ direction until it reaches RNAP at release site, then releasing RNAP from template by smacking it off (yo-yo)  - Transcription terminator: segment of DNA that signals where transcription must end. Transcription doesn’t actually end until this segment is fully coded, meaning it ends up being transcribed. (Becomes part of 3’ UTR) Video: https://www.youtube.com/watch?v=ot075b_LLHg Random expression info (entire unit explained below)  - Activators/repressors in transcription initiation: repressors block RNAP access to key promoter elements, so closed complex can not be achieved. Activators help recruit RNAP to promoter, position sigma factor to easily drive open complex formation - Sigma factor with RNAP permits promoter recognition and binding of RNAP to promoter. Transcription factors regulate transcription by activating or repressing transcription of specific genes Cotranscription - As soon as translation beings, ribosome already begins to translate. Complex of translation and transcript occurs simultaneously, called “expressesome.” Ribosome binds to Shine-Dalgarno sequence. This sequence is in the 5’ UTR Genetic Code Qualities - Triplet code, each codon is composed of three bases - Non-overlapping, every coding base on mRNA will only be included in one codon - Specific, each codon will only identify one amino acid - Degenerate, several amino acids that are coded for by more than one codon - Consists of start and stop signals (1 start & 3 stop codons) - Practically universal, majority of eukaryotes have the same codon profile Translation Translation overview - Begins with initiation (formation of 30S PIC, then 30S IC, 70S IC). Then elongation (decoding, peptide bond formation, translocation). Finally termination Translation Initiation - 30S PIC initiation: initiation factors IF3 and IF2 bind to 30S subunit, followed by IF1. Initiator tRNA (+ fMet) recruited by IF2, IF3 prevents association with 50S subunit and ensures proper selection of initiator tRNA (+ fMet). IF1 enhances activities of IF2 and IF3. - 30S IC initiation: mRNA is recruited and codon recognition starts, converts 30S PIC into initiation complex  - 70S IC initiation: 50S subunit joins and triggers dissociation of initiation factors (IFs) and places initiator tRNA (+ fMet) in P site of 50S. This is 70S initiation complex (70s IC), and is ready for translation elongation Video: https://www.youtube.com/watch?v=ch86Ny2ghhI Translation elongation - Achieved by repetitive cycles equal to number of amino acids of polypeptide, consisting of decoding, peptide bond formation, and translocation. - Decoding: ribosome translates sequence of codon from mRNA into what amino acid it needs to add to polypeptide. Ribosome chooses the complementary aa-tRNA, is delivered to ribosome from EF-tu protein - Peptide formation: ribosome has peptidyl transferase region, which promotes formation of a peptide bond between peptidyl-tRNA in P site and next aa-tRNA in A site - Translocation: promoted by EF-G, ribosome “moves over” one codon along mRNA in 5’ to 3’ direction. tRNAs in P and A sites remain attached, allowing tRNA at P site to move to E site and tRNA at A site to move to P site, leaving A site open to receive next aa-tRNA for next codon. Growing peptide moves through polypeptide exit tunnel of ribosome. Note: EF-Ts recycles EF-Tu after it delivers an aa-tRNA so it can be recycled into the pool of aa-tRNA Translation termination - Ribosome encounters stop codon, three steps follow. Recognition of stop codon, hydrolysis of ester bond on peptidyl-tRNA (by RF1/RF2). Dissociation of RF1/RF2 from ribosome with help of RF3. RRF and EF-G help separate 30S from 50S. Last tRNA is dissociated by IF3 Video (not that great): https://www.youtube.com/watch?v=TkXEl9usLN4 Protein Structure and Modification Structure - Primary structure, amino acids making up peptide. Secondary structure, alpha helices and beta sheets from interaction between amino acid R groups. Tertiary structure, 3 dimension takes into effect. Quaternary structure, two or more peptides form specific functional protein Modification - modifications made to the side chains of amino acids, can be irreversibly or reversible modification. Change the final protein conformation and thus function Genes - Eukaryote (vs Prokaryote) Eukaryotic genes: has upstream regulator sequence (made up of enhancers/silencers distally and promoters proximally like TATA). Has Open Reading Frame (ORF) (made up of exons and introns, with a poly-A signal sequence). Has downstream regulatory sequence (made up of a terminator proximally, and enhancers/silencers distally). Main differences between Euk. & Prok. Euk. Transcription influenced by - Internal cellular signals, external/environmental signals, complex interplay. Inter cellular signals are DNA binding factors, chromatin state, transcription machinery. All three of these exists to properly manage different tissues and developmental stages and better response to homeostasis of a larger organism in general Gene expression Gene structure - Euk. have enhancers that are recognized and bound by tissue-specific transcription factors. Euk. have enhancers/silencers and multiple promoters, introns, poly-A signal, and downstream enhancers/silencers mRNA structure - Both have 5’ UTR 3’ UTR, ribosomal binding, both prevents degradation. Euk are cistronic, 5’ m7G cap, poly A tail (3’ end processing), and removed introns (splicing). Euk. Has mRNA modifications. Transcription Transcription initiation - Core promoter: contains transcription start site (TSS). Can bind general transcription factors and can weakly initiate transcription. Recognized by RNAP 2. TATA box in promoter - Proximal/distal promoter: found much farther away from TSS, modulates rate of transcription through enhancers, insulators, silencers - Enhancers: recognized and bound by tissue specific transcription factors. Can increase transcription of genes, often within the same TAD. Can be close or far from TSS. When far, must be mediated by mediator and cohesin proteins - Mediator proteins: act as bridge between different transcription factors  - Silencers: can recruit repressor proteins and inhibit transcription of one or more genes - NOTE: Exon 1 includes 5’ UTR before start codon. Last exon has 3’ UTR after stop codon, which includes Poly A tail Transcription termination - Determined by RNA processing, not regulated by DNA sequence level like E. coli. 3’ end RNA processing machinery includes cleavage and polyadenylation specificity factor (CPSF), which recognizes the polyadenylation signal (PAS) on RNA and has endonuclease activity. Proceeds as follows; transcription of PAS allows assembly of 3’ processing machinery, including CPSF (PAS is subunit of CPSF) and other proteins, endonuclease and phosphatase domains of CPSF become active cleaving the transcript about 10-30nt downstream of PAS and slowing RNAP 2 and making it termination prone. Poly A polymerase (PAP) synthesizes the poly A tail. Cleavage at PAS allows contact of RNA with 5’ to 3’ exonuclease (XRN2) which digests RNA, catching up with RNAPII and terminating transcription. PAP attaches to 3’ of mRNA. XRN2 attaches to 5’ of still being produced mRNA Video (also not great): https://www.youtube.com/watch?v=BOjPlSynthU mRNA processing Standard mRNA modification include 5’ capping, splicing, 3’ end processing - 5’ Capping: soon after transcription begins, 7-methylguanosine (m7G) covalently bonds to first 5’ nt of transcripts, protects from exonuclease attack, facilitates transport, splicing, and binding to 40S subunit - Splicing: primary transcript has introns that must be removed, and exons that must be put together. Splice junction between the two is recognized by spliceosome, a large RNA-protein complex. Once spliced, first exon will have 5’ UTR, last exon will have 3’ UTR. Start is found immediately after 5’ UTR, stop is right before 3’ UTR - 3’ end processing: ends undergo cleavage downstream of PAS. PAP synthesizes poly A tail by adding about 200 adenine residues at 3’ end of cleaved transcript, helping in transport and binding to ribosome Alternative splicing - memorize Isoforms results of alternative splicing. Can produce distinct proteins Alternative splicing mechanisms - Exon skipping: meaning one or more exons may be excluded from final mRNA, some can be either included or excluded - call cassette exon - Alternative 5’ and 3’ splice sites: exons can be shortened or lengthened by using alternative 5’ and 3’ splice sites - Mutually exclusive exons: gene may include multiple cassette exons, which can be mutually exclusive so only one of them will be included in mature mRNA while others are splice out.  - Intron retention: caused by failure to remove and will be in mature mRNA - Alternative promoter: different tissues with particular transcription factors will facilitate use of one or more alternative promoter, allowing for transcription at different sites - Alternative polyadenylation: last exon may be longer or short by PAS, generating sequence diversity at 3’ end of mRNA Effect of alternative splicing impacts peptide sequence changes protein conformation. Specific isoforms may be required at different development stages or specific tissues/cells. Splicing errors can cause variety of conditions/diseases Translation-meh Extremely similar. Here are difference: - Ribosome is larger - Initiation is highly regulated comparatively - More transcription and mediating factors Mutations Mutated vs normal allele - Allele refers to sequence variation, which are usually neutral to the organism. Mutated alleles are specifically when that sequence variation causes disease or specific conditions. Distinguished between normal allelic variation in population (wild type allele), and alleles that produce alternate phenotypes are mutant alleles - is specific to population studies Reasons for study - Mutation is a part of evolution - Mutations are a source of disease, and when studied help understand pathology of big diseases - Mutagenesis is where mutation is utilized against various models to understand gene expression and regulation Mutation classification Missense mutation - Mutation leads to modification of one coding codon. - “Hypomorphic variant” Missense can impact genes essential for development. Individuals with abnormal development are often compound heterozygotes, two different hypomorphic variants. It is suspected that minimum threshold of protein activity is required for normal development - Can impact protein folding, specifically alpha helices and beta sheets and formation of disulfide bridges Neutral mutation (conservative substitution) - Mutant amino acid may have similar properties to original, no measurable impact on protein function/phenotype of individual. Silent mutation - Alteration of DNA sequence where mRNA produces synonymous codon, where sequence and structure of protein is unchanged. CAN however alter the efficiency of production. Nonsense mutations (non conservative substitution) - Base substitution or indel produces early stop codon on mRNA, producing truncated protein or no protein at all - Nonsense mediated decay: cell recognizes the EJC left by nonsense mutation and causes cell to destroy. EJC referenced in eukaryotic cotranscription Types of mutations Point mutation - Only one nucleotide is modified Chromosomal rearrangements  - Large DNA segments in one or more chromosomes are shuffled Base substitution - Mutation where only one base pair of sequence has been modified. When substitution produces change of unknown consequence, called SNP, or single nucleotide polymorphism. Can be of two types, transition where purine is traded for other purine or pyrimidine for another pyrimidine. Or transversion where purine is traded for pyrimidine or pyrimidine for purine Indels - Mutation involving 1bp to several Mb of genomic sequence. Includes deletions of one or more nucleotides, or insertion of one or more nucleotides In-frame/frameshift mutations - Indel of a protein coding region. In frame indel causes the deletion or addition of a single codon (3bp). Frameshift is not a multiple of 3 and causes the frame of each codon being read to shift, altering the entirety of the following amino acid sequence Dynamic mutations - Indel that can involve STRs w/ units of 3-6 nt. Can become unstable, adding/deleting multiple tandem repeats during cell division. Diseases caused by this show anticipation, phenomenon where diseases appears earlier with greater severity in subsequent generations. - Examples: Huntingtons; STR is within a gene, number of repeats determines whether or not toxic proteins form and aggregate in cell, due to overproduction of specific amino acid of protein leading to misfolding. Fragile X syndrome; STR in 5’ UTR, same idea as huntingtons with number of specific repeats determining pathogenicity, but instead it doesn’t code for amino acid. Instead, it causes hypermethylation of the region. Produces highly stable hairpins that causes mRNA to not be able to be degraded - alters cell homeostasis Transposition - Transposon is inserted into a new locus Loss/gain of function mutations Way mutations are classified. - Loss/gain is attributed to the change of gene expression, or the function of a protein (usually about one/more domain). Loss - Means mutations cause inactive regulatory sequence, elimination of protein synthesis, or produce protein with reduced/no function. Two types, knockout mutation when sequence/protein shows complete loss of function, or knockdown where function is reduced, or amount of protein produced is insufficient to produce normal phenotype. Gain - Rarer and it is where protein can acquire new function, is usually detrimental and occurs on one or more protein domain. Loss examples - Loss example in regulatory sequences/RNAP. Can affect promoter identification, TSS identification, or aggregation of pieces. In all ways, transcription is halted - Loss example in enhancer. Enhancer erased, certain genes will not be transcribed. Gain/loss example is rearrangement, where enhancer moves to a new location and can turn on a gene that was previously not transcribed - Loss example in splicing. Loss of splicing acceptor, exon can be removed. Gain/loss of function would be activation of new splicing site where previous intron part is transcribed - Loss/gain example in recognition of start codon. 5’ UTR can have mutation that pulls ribosome into transcription (usually makes small ribosome, can also prevent the transcription of the protein of the rest of the downstream default mRNA protein) This is leader protein. Kozak is mutation in 5’ UTR that doesn’t allow binding of ribosome. Mutation of code that causes hairpin that effects downstream transcription Gain explanations - Missense can cause enzymes to act on new substrate - Genomic rearrangements can cause gene duplication, increasing protein production - INDEL can alter TAD limits/location of regulatory elements - CHimeric genes can be formed by fusion of 5’ end of one gene and 3’ end of another - can be caused by deletion, inversions, or translocation Genomic rearrangements Alterations involving segments of DNA containing one or more genes and or regulatory sequences. This is seen in diploid organisms. When it effects phenotype, called genomic disorder. Can cause copy number or structural variants. Often caused by repetitive sequence architecture. Includes duplication, deletion, insertion, inversion, or translocation Inversion - Requires two double strand breaks on a chromosome and segment must be reinstated to the same chromosome, but opposite orientation. Pericentric inversion involved both arms of chromosome and centromere. Paracentric inversion only involves one chromosome arm, no centromere Translocation - Exchange of DNA segments between two non-homologous chromosomes. They can be balanced, when no genetic material is lost at the break points, or unbalanced, when they do - Robertsonian translocation: centromeric fusion of two acrocentric chromosomes, most often between non-homologous chromosomes. Remaining p arms are known to also fuse but the product i lost during cell division due to lack of centromeres/kinetochores Random vocab Aneuploidy - Loss or gain of one or more chromosomes of diploid set, usually severe Nullisomy - No copies of specific chromosome are found Monosomy - Loss of one chromosome of homologous pair Trisomy - Three copies of the same chromosome Tetrasomy - Four copies of the same chromosome Uniparental disomy - Individual has two copies from one parent, making up homologous pair Polyploidy - Multiple copies of entire set of chromosomes. If this is true for the species then its is autopolyploid. If the extra set is from a different species (mostly plants but some animals that can hybridize), it is called allopolyploid Eukaryotic Gene Expression Overview - Can be short or long term regulation that allows cells to respond rapidly to the environment and modes of living (age/stage). Important to note/know is chromatin conformation, DNA methylation, noncoding RNAs, X-inactivation, transcription regulation in initiation, transcription regulation in elongation & termination, post transcription regulation, and regulation of translation. Most have to do with promoter availability Methods of gene expression regulation Chromatin conformation: Transcription requires access to the promoter, which chromatin must be in a relaxed state to allow - Chromosome territories and mini territories within which are associated with active or repressed DNA sequences (location of chromosomal segment in nucleus) - Interphase chromosomes are organized into TADs, which are active/repressed distinct by cell types (organization of chromosome in TAD) - Existence of euchromatin and heterochromatin, controlled by level of H1 association - Histone tail modifications (epigenetic code) produce variety of chromatin states associated with different levels of transcription, among other chromatin binding proteins - ncRNAs can bind DNA and proteins that bind DNA/RNA, found in scaffolding complexes that impact chromatin structure, can recruit chromatin remodeling complexes Epigenetic profile and imprinting - DNA methylation - C bases of CpG dinucleotides can be methylated to produce 5-methylcytosine (5-meC). Methylated DNA attracts proteins that bind to it, which can either be or call more proteins to bind - usually is transcription repression as transcription factors can’t bind to methylated DNA. Changes chromatin organization, transcription, and can cause extra epigenetic regulation. CpG islands are usually not methylated. - Heritability of DNA methylation - 5-meC is copied to complementary strand through enzyme, allowing heritability of methylation. Gametes go through wave of demethylation during gametogenesis, will then be methylated at specific loci following pattern based on parent of origin (heritability), called differentially methylated region (DMR). Most epigenetic marks persist during mitosis but not meiosis, methylation is the exception. Demethylation is important since the whole code must be copied many times, need access to the DNA. - X-inactivation - Females have two X chromosomes, men have one. Y chromosome only houses male development and fertility related genes (little), while X chromosomes hold essential genes. Female X chromosomes can not exist as two together, so X-inactivation must occur. - Has a locus called X inactivation center (XIC). During embryogenesis, paternal & maternal X chromosomes line up, one to be inactivated synthesizes large non coding RNA, called X-inactivation specific transcript (XIST), is processed and binds to X chromosome, recruiting other repressive proteins. - Dosage compensation: With two X chromosomes, females would translate double the amount of required products, instead - X is inactivated - Most genes will be constitutive heterochromatin. Each cell of blastula randomly chooses maternal or paternal X chromosome to inactivate, for females. This means cells within a single individual can differ from each other, see cat example below. 15% can be accessed on inactivated X chromosome, on Pseudoautosomal region (at the top and bottom - called PAR) Transcription regulation in initiation: Think about promoter availability, and RNA pol II being able to go to elongation mode - Promoter regions must be devoid of nucleosomes for transcription factors, with downstream having loosely packed nucleosomes - Transcription factors may recruit additional proteins important for co-activation/repression, impacting accessibility of promoters and regulatory sequences - Promoters can be bound by pre-initiation complex (PIC) even if downstream is not being expressed, most likely that gene expression is regulated by how efficiently RNA pol II can begin elongation, rather than if PIC can assemble - Alternative promoters can exist which can lead to protein variation as seen in alternative splicing - Enhancers require DNA looping, which occurs in part due to the structure of the TAD is exists in. Changes in the TAD can alter enhancers ability to reach promoters - Antisense and bi-directional transcription of ncRNAs interferes with sense transcription, DNA methylation, modifying histone code, inducing heterochromatic states, silencing transposons  - Waddington’s epigenetic landscape is the fact that in early development, master transcription factors bind to super-enhancers to shape and define early cellular identity, with a much later in life stage and established cellular identity being controlled with very fine tuned expression and limited regulatory sequences bound by regulatory proteins Post-transcriptional regulation - Alternative splicing leads to isoforms - Important for different tissues have different proteins through expression change - Especially important for neurons, creating ion channels and receptor variants - Enhancers and suppressors can be spliced, changing expression of genes - Can cause poison exons (premature stop codon), causing nonsense mediated decay - RNA turnover (abundance and availability) and RNA editing Regulation of translation - Leader peptides - Structure of mRNA: can have stem loop structures that impact ribosomes ability to move forward, sites can be more open to RNA binding regulatory proteins - ncRNAs: miRNA siRNA piRNA together can repress expression of target mRNAs. miRNA can also bind 3’ UTR to modulate rate of translation and can compete for the target mRNA Prokaryotic Gene Expression Occurs through transcriptional regulation primarily and translation regulation Operon and regulation Operon - Group of genes under control of a common promoter and regulatory elements that produce a single and polycistronic mRNA - Inducible operon; encodes group of enzymes involved in catabolic pathway, seen if the presence of particular substrate stimulates transcription - Repressible operon; encode group of enzymes involved in anabolic pathway, seen if the presence of particular product inhibits transcription Regulation of operons - Availability of sigma factor and RNAP - Regulators (transcription factors) bind to DNA binding sites and can repress/enhance transcription - Can be activators; work by binding DNA of the promoter and and enhances transcription (positive control) - Class 1 activation; transcription factor binds upstream of core promoter sequences, interacts with alpha subunit of RNAP and promotes sigma factor binding - Class 2 activation; transcription factors bind very close to core promoter and facilitates sigma factor binding - Dna conformation change; DNA helix at the core promoter is twisted to facilitate sigma factor binding - Repressor modulation/anti repression; prevents repressor from binding DNA or its capacity to impede transcription - Can be repressors; work by binding to parts of the DNA within operon (negative control) - Steric hindrance; repressor binds to core promoter elements or between them, obstructing RNAP binds (lacI) - Roadblock; transcription elongation cannot progress as repressor binds to the start of the coding region - DNA looping; repressor binds upstream and downstream of core promoter and the monomers interact, causing the DNA to loop and impedes RNAP binding - Modulation of activator; repressor binds to DNA element that partly overlaps with activators binding site, preventing the binding of the activator  - Attenuation is where the regulator targets RNAP or the actively being made transcript. Either pauses or terminates transcription to modulate expression of structural gene of the operon - Antitermination is where regulator allows transcription to proceed past initial termination signal (where the default pathway would lead to premature termination of transcription). Early termination or the default pathway only occurs when regulator is not bound to the RNAP Examples to memorize Important to remember that for prokaryotes, transcription/translation occurs cotranscriptionally lac Operon (https://www.youtube.com/watch?v=LuOaEe89_HE) - Gene function: allows E.coli to metabolize lactose. Negative inducible operon - Made up of: promoter (lacP), operator (lacO), lacZ, lacY, lacA, and terminator, and upstream regulatory gene with a promoter and lacI gene - Each genes role: - lacZ: produces beta-galactosidase, an enzyme that breaks down lactose to glucose and galactose - lacY: produces permease, transmembrane protein that allows lactose to enter the cell (disaccharide too large to diffuse) - lacA: produced galactoside acetyltransferase, function unsure - Regulatory gene: lacI found upstream, produces a repressor - When lactose is absent (glucose present): lacI produces regulator protein, it binds to lacO, blocking RNAP. Genes needed for lactose metabolism are not transcribed/translated. Energy needs are met since glucose is present, no need/ability to metabolize lactose - When lactose is present (glucose absent): lacI produces regulator protein, lactose and allolactose enter the cell, allolactose binds and inactivates repressor protein. LacO is available for binding, and lac genes are transcribed/translated. Cells need energy source, low glucose means lactose should be metabolized, so expression of lac operon is allowed - When lactose is present, allolactose (a slightly different form of lactose), deactivates the repressor, allowing for the enzymes need to metabolize lactose to be translated - Carbon catabolite repression (positive control): If there is low glucose levels, there will be high cAMP levels, cAMP binds to CAP which facilitates RNAP binding to lacP. If high glucose, low cAMP, cAMP doesn’t bind to CAP, which won’t facilitate RNAP binding to lacP  Lactose present Lactose present Lactose present Lactose absent Lactose absent Lactose absent B-gal Perm Trans B-gal Perm Trans LacI+ lacP+ lacO+ lacZ+ lacY+ + + + - - - LacI+ lacP+ lacO+ lacZ- lacY+ - + + - + + LacI+ lacP- lacO+ lacZ+ lacY- - - - - - - LacI+ lacP+ lacO+ lacZ- lacY- - - + - - + LacI- lacP+ lacO+ lacZ+ lacY+ + + + + + + trp Operon (she didn't explain the WHY so i just couldn’t understand, this is super helpful but jam packed so i had to rewatch it a couple times: https://www.youtube.com/watch?app=desktop&v=CeE83RyQFRo) - Gene function: allows E. coli to synthesize triptophan (through transcription attenuation). Negative repressible (considering trpR). Attenuation for structures - Made up of: promoter, operator, trpE, trpD, trpC, trpB, trpA, terminator, and upstream regulatory gene with a promoter and trpR gene. Has a leader peptide region (in 5’UTR) that is split into 4 regions - Genes role: are set of enzymes that convert chorismate to triptophan. trpR gene produces active repressor (W) - Pause structure is combination of region 1 and 2, a loop that stalls RNAP, giving ribosome time to initiate translation of leader peptide. Ribosome catches up to RNAP, transcription resumes and the two processes continues on to either 2-3 or 3-4 region - Antiterminator structure is combination of region 2 and 3. Acts as an antiterminator, signaling RNAP to continue and transcribe all structural genes. Occurs due to a codon in the 1st region requiring triptophan for translation, there isn’t enough free triptophan so ribosome stalls, stalling allows the 2nd and 3rd region to form a loop. 2-3 signals RNAP to transcribe structural genes, ribosome eventually translates the mRNA and enzymes are produced - Intrinsic transcription terminator sequence is the combination of region 3 and 4, terminates transcription before RNAP reaches the structural genes. Occurs because triptophan level is high, and ribosome does not stall in 1st region. Ribosome encounters a stop codon it usually wouldn’t because RNAP did not have enough time to move forward, transcription halts, and structural genes are not transcribed - Additional mechanism: If triptophan concentration is too high, trpR (inactive repressor) allows for binding of triptophan which activates its ability to impede binding of RNAP to trp promoter, transcription does not occur Basic operon vocab - Pos-inducible: In the presence of a substrate, activator is active, transcription on - Neg-inducible: In the presence of a substrate, repressor is inactive, transcription on - Pos-repressible: In the presence of a product, activator is inactive, transcription is off - Neg-repressible: In the presence of a product, repressor is active, transcription is off Non-operon regulation Genome/chromatin structure: - Shape can change availability of RNAP to bind - Influenced by NAPs and lncRNAs - NAPs can modify chromosome architecture, mediate interactions between ncRNAs, moderate transcription by blocking promoter, advancement of RNAP, supercoiling, and binding factors Epigenetic profile: - Methylation alters availability of RNAP to bind - Restriction modification (R-M) - To remove unwanted DNA within bacteria, have endonucleases. To protect own chromosome against endonucleases, can have endogenous methylases - Orphan methylases (GATC sites) regulate; gene expression: methylation allowing binding of activators or repressors, DNA repair: methyl-directed mismatch repair (MMR) can recognize template strand, initiation of DNA replication; methylation at promoter of dnaA and oriC allows binding of replication initiation regulator protein (SeqA) to prevent unwanted reinitiation Transcription regulation: - Is operon regulation, so above Post-transcriptional regulation: - RNA editing: rNTPs are modified after transcription and can alter amino acid sequence - RNA turnover: amount of RNA and availability is balanced by abundance/location of RNases, and binding to ncRNAs and others Translation regulation: mRNA and protein abundance correlates but weakly, as protein synthesis is very energetically demanding, so mechanisms control translation rates. Include:  - Formation of secondary structures in mRNA - Presence/strength of different ribosome binding sites (RBS) on the mRNA - Codon usage bias where specific codons are used more than others - Leader peptides which terminate transcription before RNAP transcribes first structural gene of operon (like trp) - Riboswitches found in 5’ UTR most often that respond to molecules/ion ligands that bind and alter folding of mRNA Regulation via non-coding RNAs: - Can degrade and alter expression of mRNAs - Cis encoded ncRNA elements: structures found in 5’ UTR, responds to cellular or environmental changes. Like attenuation, leader peptides, riboswitches - Cis encoded antisense RNAs: target mRNA for degradation, encoded in or close to gene but produced from an antisense promoter - Trans-encoded antisense ncRNAs: target mRNA and impacts transcription and translation, found in different locus from gene they regulate Molecular Cloning Purpose: used to amplify large DNA segments too large for PCR, or to study protein encoded by specific genes Parts of technique Gene of interest - Gene to be amplified or studied is cut and placed into the vector - Requirements: - Has restriction sites that match vector, that flank the gene of interest Vector - Plasmid that is inserted into bacterial cells - Requirements: - Has an origin of replication - Has selectable markers (for our purpose this why ALWAYS be lacZ and may be some sort of antibiotic resistance like ampR gene) - Has unique restriction sites on lacZ Construct - Combination of vector and gene of interest - Basically will be the vector (plasmid with antibiotic resistance and lacZ) hybridized with the gene of interest Competent E. coli - E. coli that the construct is absorbed by must already be competent, meaning already has a lacZ gene, see in reading screening results Steps of cloning - Choosing restriction site: choose restriction site that flank the gene of interest and exist within the lacZ gene of the vector - Insert/vector preparation: enzyme specific to chosen restriction sites are mixed with the gene of interest and plasmids separately. Restriction enzymes cut palindromic sequence of the restriction site, leaving 3’/5’ overhang or “stick ends” for next step - Ligation: vector and gene of interest are mixed together with ligase, which stitches the bonds between the two (called hybridization) - Bacterial transformation: the plasmids are inserted into a bacterial cell colony (which can take up free plasmids) - Colony screening: agar plate for growing bacteria includes Xgal and ampicillin (or other antibiotic). Colonies exposed to the constructs are placed on the plate for growth, results are read - Orientation test: ligation could have hybridized the gene of interest towards the promoter or away from (forward or reverse respectively). PCR and gel electrophoresis help reveal which orientation they were hybridized. First, we must choose a new restriction site. The original isolation of the gene of interest left some extra DNA upstream and downstream of the gene - on this area there MUST be at least one new restriction site. This same restriction site must exist on the vector, pre-insertion of gene of interest. For the example picture provided, the vector originally had 3 SmaI restriction sites. With our insert, it would total 4. PCR and gel electrophoresis after cutting SmaI of a white colony would present 4 different strands of DNA of different bp readings. If by comparing the gel electrophoresis results (4 reading of different bps) with the theoretical bps of the white colony cells between SmaI restriction sites matches, the orientation is forward. If they do not match, it is reverse orientation. Example provided. Reading screening results - There are three general colony outcomes. Colonies that did not survive, colonies that survived and are blue, and colonies that survived and are white. - Colonies that did not survive: Because there is an antibiotic on the plate, if the plasmid was not absorbed by the bacterial cell, it would not survive. If a bacteria did absorb a plasmid, but the gene of interest inserted into the resistance gene, it would not produce functional mRNA and would lose its resistance and die. If ligation was not achieved, the bacterial cell could have received an unligated DNA (which would be linear DNA) which is not function - or it could have received only the gene of interest, and die. - Blue colonies: the plasmid was successfully received and ligated, as the resistance gene is being expressed. However, the vector does not have the proper insertion of the gene, either not hybridization was achieved, or the gene of interest was inserted into the wrong area. The reason that they are blue is because of the Xgal on the plate being a lactose analog - the lacZ gene converts this analog into galactose which is blue (from beta-galactosidase enzyme produced from lacZ gene)  - White colonies: a vector was properly hybridized with the gene of interest, in the correct location, and was absorbed by the bacterial cell. We know it received a functional vector because it is alive and must have a functional ampR gene, and we know it received a functional construct because the gene of interest MUST have inserted INTO the lacZ gene and disrupted it, causing the lack of beta-galactosidase, meaning they could not convert the Xgal into galactose which gives the blue color Advanced cloning random stuff - For eukaryotes that have introns, need to get rid of it, can use reverse transcriptase - If there aren’t any selectable restriction sites for orientation, can modify 5’ end of DNA by using PCR, designing primers that DO have selectable markers (on gene of interest) - The reverse orientation DOES make a different type of protein - Each colony on the plate stems from a single bacteria, will be identical per colony - Be able to draw forward and reverse orientation constructs - Differences in techniques from pork. And euk.: going from one or another, the mechanisms of using proteins, or just the promoter regulation of a DNA sequences, could cause toxicity or non functional proteins or mechanisms