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BIO 1A03 McMaster University Test 2
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Translation - ✔✔Cellular components are able to read the genetic message in mRNA and translate the message into specific primary amino acid sequence of a protein How does tRNA structure allow for SPECIFICTY in translation? - ✔✔
- Plays critical role in ENABLING the translation of the information
- Each type of tRNA molecule is not identical and can translate a specific codon into a specific amino acid What is the structure of tRNA? - ✔✔- single RNA strand (70-90 nucleotide length)
- four double helical segments and three characteristic loops How are the four double helical segments and three loops in tRNA molecules formed? - ✔✔Formed due to the large degree of complementarity along stretches of tRNA which results in many stretches of hydrogen bonding between complementary bases. What are anticodons? In what direction are they conventionally written? - ✔✔Three-base sequence in a transfer RNA molecule base that pairs with a complementary codon in mRNA They are conventionally written in the 3' to 5' direction What is at the 3' end on tRNA? - ✔✔There is a protruding amino acid attachment site made up of CCA nucleotide sequence (terminal "A" is point of attachment for amino acid during tRNA molecule activation) How is the activation of a tRNA molecule with a specific amino acid carried out? - ✔✔Activation is carried out by a family of enzymes called aminoacyl tRNA synthetase
True or False; Each enzyme is specific to the type of tRNA and corresponding amino acid it will bind. - ✔✔True - 20 aminoacyl tRNA synthetase How do aminoacyl tRNA synthetases work? - ✔✔The active site of the enzyme recognizes the anticodon end of the tRNA and the region of the amino acid attachment site. Once bound to active site, enzymes catalyze covalent attachment of tRNA molecule to its amino acid (energy from ATP Hydrolysis) Charged tRNA - ✔✔able to deliver amino acids to growing polypeptide chain. What does correct translation require (in terms of codon and anti-codon)? - ✔✔Correct translation requires the correct PAIRING of tRNA anticodon with the appropriate mRNA codon How does codon-anticodon pairing occur? - ✔✔The 5' end of codon (mRNA) will bind with the 3' end of the anticodon (tRNA) What is meant by the term wobble? What does it help to explain? - ✔✔Greater flexibility for base pairing between third nucleotide of a codon and corresponding base of tRNA anticodon Wobble helps explain the redundancy of genetic code When does translation initiation in Eukaryotes occur? - ✔✔In eukaryotes, it occurs when a translational initiation complex forms towards the 5' cap of the mRNA and then scans the mRNA until an AUG start codon is encountered How does translation initiation in Prokaryotes occur? - ✔✔Since there are no 5' caps, translation initiation complex will assemble at one or more ribosome binding sites called Shine-Dalgarno sequences (Located a few bases upstream the translation start codon)
How are peptide bonds formed in translation? - ✔✔Once tRNA binds mRNA, a conformational change occurs that is induced in ribosomal RNA that allows for peptidyl-transferase reaction to occur The condensation reaction occurs as peptide bond and transfers amino acid to growing polypeptide chain onto tRNA in A-site When another tRNA binds the site, what happens? - ✔✔The E-site is ejected How is translation terminated? - ✔✔Once ribosome reaches stop codon, GTP-bound release factors will bind A-site and catalyze hydrolysis of bond between terminal amino acid in polypeptide and tRNA in P- site Further GTP hydrolysis will also enable the dissociation of the translation complex one gene-one enzyme hypothesis - ✔✔the function of a gene is to dictate the production of a specific enzyme
- Based on the fact that Neurospora can grow well on minimal medium
- They must have some enzymes produced by a specific gene that convert simple substances into amino acids and vitamins necessary for growth Neurospora are able to synthesize what? - ✔✔Able to synthesize arginine through metabolic pathway What did Srb and Horowitz demonstrate? - ✔✔Each gene contains the information needed to make each enzyme How did Srb and Horowitz test their hypothesis? - ✔✔- Performed genetic screen of radiation treated neurospora to determine if there are specific enzymes produced in the metabolic buildup of arginine
- Radiation would lead to mutations so they conducted the genetic screen by raising colonies of radiation treated cells on a medium that was supplemented with nothing, ornithine, citrulline, or arginine added to medium
What was the result of Horowitz and Srb's experiment? - ✔✔1. With supplemented arginine, fungus is able to undergo growth
- Non-supplemented medium, fungus not able to grow
- Orthinine and Citrulline only: Inhibition in growth Identified 3 mutants: Arg 1 (grows in all), Arg 2 (Citrulline and arginine only), Arg 3 (arginine growth only) What is the exception to the one-gene, one enzyme hypothesis? - ✔✔- 20 - 25,000 protein encoding genes in human genome
- Provided evidence that more than one protein can possibly be produced from a single gene
- Alternative splicing + other mechanisms contribute to complexity of mRNA and therefore protein The Human Proteome - ✔✔The full number of proteins that are expressed by all the hereditary information in our DNA. (20-25,000 protein encoding genes) The complexity of our proteome relative to our genome is attributed to what? - ✔✔The great complexity is largely attributed to RNA processing and post translational modifications True or False, the composition of proteome can change in response to various factors? - ✔✔True - Occurs in organisms developmental stage and in response to internal and external signals How does a cells ability to detect changes in environment benefit the cell? - ✔✔Changes serve as stimuli that will result in important cellular responses What happens in your body after a meal? - ✔✔1. Stimulus can include an increase in Blood glucose levels (regulated by sensory responses in specialized islet cells of pancreas)
- Will lead to a cascade of events that return blood glucose back to normal What happens in the case of high blood glucose? - ✔✔The pancreas will modulate the synthesis and secretion of an increased amount of its own signal, insulin
Once proinsulin is folded, where is it transported? - ✔✔Transported from RER to golgi apparatus where further cleavage occurs forming mature insulin dimer (releases C chain) Phosphorylation - ✔✔Reversible modification that involves the covalent attachment of a phosphate group to serine, threonine, or tyrosine residues in a protein by enzymes called kinases Methylation - ✔✔Covalent addition of methyl groups to DNA strand (Particularly promoter) Acetylation - ✔✔The addition of acetyl groups to specific amino acids Receptors - ✔✔Important proteins that receive and interpret information from such signalling molecules like insulin
- Each receptor has the ability to bind specific signals What are receptor kinases and what is their purpose? - ✔✔Insulin protein binds specific insulin receptor that fall into a family of receptors called receptor kinases (Binding of insulin to receptors enables many cells to transport glucose across plasma into cytosol) Binding of insulin to these receptors causes a conformational change which causes the receptor monomers to pair up, leading to the activation of cytoplasmic domains of the receptor which have the ability to act like kinase proteins Kinase proteins - ✔✔Able to engage in phosphorylation of specific amino acids What occurs as a result of intracellular signal amplification? - ✔✔Extracellular insulin signal causes a series of cytoplasmic proteins to become sequentially activated and will lead to an intracellular response which is the activation of GLUCOSE TRANSPORTER PROTEINS at cell surface to increase absorption of glucose into cell How are intracellular signals induced? - ✔✔Once insulin binds to receptor, intracellular signals are induced through the activation of a series of diverse TRANSDUCER AND AMPLIFIER PROTEINS that are downstream from activated receptor
Double negative feedback - ✔✔Inhibitor of the signal can also be inhibited To produce more than one mRNA transcript from a single protein coding gene, what must occur? - ✔✔Alternative splicing of pre-mRNA's must occur Alternative splicing - ✔✔Enables one pre-mRNA molecule to be spliced at different junctions to result in many different mature mRNA molecules that each contain different combinations of transcribed exons Why might some exons be excluded during the splicing process? (This leads to the production of many isoforms of different types of mature mRNA from same pre-mRNA transcript. - ✔✔This occurs because what the spliceosome will sometimes recognize as an exon in some primary transcripts, can sometimes be identified as an intron in other primary transcripts What occurs in skeletal muscle cells that makes them have a higher affinity to insulin compared to liver cells - ✔✔In skeletal muscle cells, Exon 11 is removed from mature mRNA product of gene coding for insulin receptor. In liver cells, exon 11 is attained which is why it has a lower affinity How is the signal for insulin terminated? - ✔✔After body cells have taken up glucose, there is a decrease in blood glucose that will be detected by pancreatic cells and there will be a decrease in the secretion of insulin
- The feedback of this information to the the sensor cells limits any further response in the entire system Under favourable conditions and available nutrients, all cells have the essential machinery that favours? - ✔✔Cell growth and division Most prokaryotes grow best at? - ✔✔Certain favourable temperatures True or false, the DNA of a bacterial nucleoid contains information required to orchestrate a response to any change in the environment - ✔✔True
True or false, it is the products of glucose metabolisms themselves that activate the switch between glucose and lactose use - ✔✔True In the absence of a direct source of glucose, the E. coli cell can metabolize the disaccharide lactose. What is lactose composed of? - ✔✔Glucose and Galactose What is the enzyme that can metabolize lactose to produce glucose and galactose called? - ✔✔Beta Galactosidase enzyme True or false, the gene that codes for B-galactosidase is constitutively expressed (always on) - ✔✔False, the cell will only transcribe the gene when lactose is present and glucose is not Francois Jacob and Jacques Monod investigated how E.coli are able to produce B-galactosidase that is needed for lactose metabolism, what did they observe? - ✔✔- observed that production of the enzyme is dependent upon presence of lactose in environment
- Observed the amount of B-galactosidase protein produces by the E.coli cells began to steadily increase in response to addition of lactose (also, production of enzyme ceased when lactose was removed) gene expression - ✔✔The functional product of the gene is made, modified and activated How many distinct levels of regulation must occur? - ✔✔Three levels - Transcriptional control, translational control, post-translational control True or false, if the transcriptional control level was disrupted, there would still be an activated protein - ✔✔False, if any one of the steps is disrupted, there will be NO activated protein Transcriptional Control - ✔✔> Controls the amount of mRNA that is produced in the cell In prokaryotes and eukaryotes, activation of transcription requires what? - ✔✔Requires that proteins bind to a region near the beginning of the gene (the promoter) and increase binding of RNA polymerase
- gene is now transcribed or turned on.
Translational Control - ✔✔gene expression regulated by influencing the interaction of the mRNA transcripts with the ribosome Occurs by binding of the ribosome to the 5' cap of mRNA True or false, stability is a characteristic that determines the amount of protein produced - ✔✔True, if mRNA is quickly degraded, very little protein is made post-translational control - ✔✔Regulation of gene expression by modification of proteins (e.g., addition of a phosphate group or sugar residues) after translation.
- Control mechanisms allow polypeptide chain to be folded into 3d Structure More than a dozen post-translational modifications regulate the ability of the protein to become active or inactive by doing what? - ✔✔Driving the assembly into complexes, binding of substrates, unmak enzymatic domains Which level of regulation is the fastest?(quick cellular responses) - ✔✔Post-Translational regulation is fastest as it allows the cell to have a stockpile of protein in the cell that is simply inactive
- Once the cell receives a signal, it can lead to a simple modification to turn on all of the inactive proteins Which level of regulation is the slowest? - ✔✔Transcriptional regulation is the slowest as the cell is starting from scratch
- Since cells must first transcribe, then translate and modify, it results in the delay of what is being produced Which level of regulation is the most efficient? - ✔✔Transcriptional regulation is most efficient as it does not waste any energy or resources making a mRNA or polypeptide unless it really needs to How can efficiency be observed in E. coli cells - ✔✔They only increase gene expression of B- galactosidase gene in presence of lactose as a nutrient supplement within their local environment They are able to efficiently metabolize lactose on an as-per-needed basis
Lac1 gene - ✔✔Controls expression of lacZ and lacY genes. How does the Lac1 gene control the expression of LacY and LacZ genes? - ✔✔It codes for a repressor protein which can bind to the operator and inhibit transcription from occurring Negative transcriptional regulation - ✔✔The ability of a repressor protein to halt transcription via a repressor What is the structure of the repressor protein? - ✔✔It is a tetrameric protein made of four identical protein subunits that binds tightly to the operator region of the lac operon Once all four subunits bind the lac operon DNA, what happens? - ✔✔DNA is twisted into a loop and RNA polymerase is not able to bind to the promoter and transcribe the gene. The presence of glucose in the bacterial cell growth medium facilitates what? - ✔✔The expression of the repressor protein How is lactose able to act as an inducer molecule? - ✔✔lactose binds to the repressor proteins on the lac operator and causes a change in the conformation of the repressor so that it can no longer bind to DNA operator sequence What does positive regulation of the lac operon mean? - ✔✔In the absence of glucose, positive regulation promotes the production of B-galactosidase and lactose permease If there is a decrease in environmental glucose levels, what happens to the cAMP levels inside the cell? - ✔✔Increase in intracellular cAMP inside bacterial cells True or false, it is the increase in cAMP levels that will contribute to the positive regulation of the lac operon - ✔✔True
When glucose concentration are high, how are cAMP levels low? - ✔✔If glucose concentrations are high, there is an inhibition of the enzyme adenylyl cyclase which catalyzes the production of cAMP from ATP - Results in low cAMP levels When glucose concentration are low, how are cAMP levels high? - ✔✔There is an increased activity in adenylyl cyclase which catalyzes production of cAMP - Results in high cAMP levels Positive regulation involves another protein called CRP or CAP (cAMP Receptor protein or Catobolite Activator protein), what are they responsible for? - ✔✔When bound by cAMP, CRP will bind to a different site on bacterial DNA called the CRP-cAMP binding site (Binds to CRP protein as an allosteric activator)
- The amount of cAMP will determine whether a transcriptional activator will bind to this site and therefore the degree of positive regulation of the lac operon What happens when cAMP binds CRP protein as an allosteric activator? - ✔✔This induces a conformational change in the shape of the transcriptional activator protein Once conformational change in the shape of the transcriptional activator protein has occurred, what happens? - ✔✔CRP-cAMP complex binds DNA at activator binding site and further activates transcription of lactose biosynthesis enzymes in the presence of lactose True or false, Lactose can repress expression of lac operon gene products while glucose can activate expression of lac operon gene - ✔✔False, Lactose Activates expression, Glucose Represses expression Early embryonic stem cells have the ability to do what? - ✔✔The ability to mature or differentiate into many different cell types Different embryonic cells will have different fates depending on what? - ✔✔Depending on signals that are exchanged and which genes are switched on or off at a specific time. Differences in gene regulation will lead to altered proteomes which contributes to distinct differences in cellular functions. True or False? - ✔✔True
- charged tails of histone allow for negatively charged DNA to wrap tightly. True of False? - ✔✔True Acetylation of histone tails does what? - ✔✔Weakens their interaction with DNA and permits some transcription factors to bind to DNA How do activator proteins play a role in acetylation? - ✔✔Activator proteins recruit coactivator enzyme histone acetyltransferase (HAT) which attaches acetyl groups to lysine amino acids along positively charged tails of histone proteins Methylation of lysine and arginine and phosphorylation of serine and threonine along the histone protein tails does what? - ✔✔May also alter chargers which alters binding to DNA that is wound around them, allowing space for transcriptional proteins to work The degree of modifications to the histone tails is part of a histone code that determines what? - ✔✔Whether transcription is activated or repressed Transcriptional factors are able to recognize and bind to nucleic acid sequences in DNA based on what? - ✔✔Structural and chemical components between proteins and DNA Why are alpha helical domains of transcription factors beneficial? - ✔✔They tend to fit nicely within major grooves of DNA due to molecular interactions between amino acids and functional groups of nitrogenous bases When a strong enough interaction is made between the transcription factor and the DNA, what happens? - ✔✔Transcription factor assumes a conformation that allows for the control of transcription
- Recruitment of other transcription factors
- RNA polymerase
- Activation of Transcription Core-promoter - ✔✔The binding site that is required for binding of RNA polymerase and associated transcription factors
TATA box Region - ✔✔Recognized by TATA-binding protein subunit of the trnascription factor TFIID BRE region - ✔✔Recognized and bound by TFIIB transcription factor Enhancer Regions - ✔✔Able to bind CELL or REGION SPECIFIC transcription factors What do enhancer regions facilitate? - ✔✔They facilitate the formation of the transcriptional complex as transcription factors bound to this region are able to interact with transcription machinery at the promoter to enhance transcription of a gene What does looping of DNA allow? - ✔✔Allows for regulatory sequences which are situated far from the transcription sites to become closely associated with transcription sights Mediator proteins - ✔✔Connects proteins bound to enhance regions with proteins bound to core promoter - able to act with RNA polymerase When silencer regions are activated by a repressor protein, what does it lead to? - ✔✔Leads to interference of general transcription factor assembly and mediator activity which are needed for binding of RNA polymerase and transcription In fetal blood cell progenitors, the chromatin is wound up around what? - ✔✔Wound up around beta globin gene to inhibit transcription Chromatin around gamma globin gene is open to allow transcription The developmental switch from gamma (fetus) to beta globin proteins (adults) is regulated by what? - ✔✔Specific transcription factors that are able to silence or repress gamma-globin gene transcription and others that are able to activate beta-globin gene transcription The most common modification to the CYTOSINE BASE is? - ✔✔The addition of a methyl group True or false, methylation occurs on every cytosine - ✔✔False, it occurs within a string of cytosine and guanine bases called a CpG island (P=phosphate in backbone between two bases)
DNA microarrays are largely based on what? - ✔✔The base-pair interactions and binding of complementary strands of nucleic acid How do microarray assays work? - ✔✔1. Set up glass slides that have tiny spots containing a known DNA sequence or gene (these act as probes to detect gene expression)
- Can use genomic DNA sequences as probes to investigate whether specific genes of interest are reanscribed and cal look at groups of genes to determine whether they are expressed in any specific manner Why are microarray useful? - ✔✔To visualize variation in gene expression at various developmental stages To identify differences in gene expression of regular and cancerous cells How is it possible to identify differences in gene expression of normal breast cells and cancerous breast cells? - ✔✔- Grow both cell types in culture
- Isolate gene products or mRNA that is transcribed by the 2 cells
- Perform microanalysis to help understand what genes are involved in the transformation of a normal cell to a cancer cell Once mRNA has been isolated from breast cancer and normal breast cells, what is done? - ✔✔- Isolated mRNA serves as template for making complementary cDNA molecules to the mRNA using a specific reverse transcriptase enzyme What occurs during reverse transcription? (microarray technique) - ✔✔Fluorescent nucleotides are utilized and become part of the newly synthesized cDNA molecule - cDNA isolated from each cell should be coloured with different fluorescent dyes so they can be identified during analysis Once cDNA is labelled, what occurs? - ✔✔Both prepared cDNA molecules from each cell can be combined in equal amounts and tested for hybridization with the single stranded DNA that is present on the chip
mixed molecules of cDNA compete for synthetic complementary DNA fragments that are distributed across all the spots of the chip How is gene activity analyzed using microarray analysis? - ✔✔If a particular gene is active, it will produce many molecules of mRNA and there will be more labelled cDNA available (observe bright spots) Ex. if spot is green, specific gene in that spot is expressed more in normal cells and there is decreased expression in breast carcinoma cells In order to stop gene expression, what should happen to mRNA? - ✔✔mRNA's should be degraded Some evidence suggests that genes can be transcribed into mRNA, but that mRNA never ends up being translated, why does this occur? - ✔✔Due to the activation of RNA interfering machinery which is activated by short, noncoding, regulatory double stranded RNA molecules MicroRna (regulatory RNA molecule) - ✔✔Transcribed from protein encoding genes using the same RNA polymerase that transcribes other RNA Form hairpin loops due to complimentary base-pairing within microRNA precursor transcripts What occurs once hairpin loops in MicroRNA are formed? - ✔✔They are processed into smaller, single stranded mRNA fragments that activate RNA interference machinery
- processed microRNA becomes incorporated as part of RNA-INDUCED SILENCING COMPLEX - When the binded structure matches an mRNA, it inhibits transcription siRNA's are similarily transcribed and processed like microRNAS, what is a main difference between the two? - ✔✔Once siRNA's bind to complementary sequence by an association with RNA induced silencing complex, they induce cleavage or cutting of target mRNA What does cleavage do? - ✔✔Destabalizes target mRNA and further contributes to post-transcriptional regulation of gene expression
