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Notes on Organisation Control of Prokaryotic and Eukaryotic Genomes Introducing genomes o Each single human chromosomes is a single molecule

e of DNA o However, there is no direct correlation between genome size and morphological complexity (the C value paradox) o While human genomes is approx 10 times bigger than a typical bacterium, the number of coding genes is less than some other prokaryotic morphologically simpler organisms o This is because we have enormous amounts of non-coding DNA o Therefore, in order for such a relatively small difference in the number of genes (C value) to generate such morphologically striking differences must be due to other factors Chromosomal rearrangement Alternative splicing to produce multiple mRNAs Variations in post-transcriptional modification Describe the structure and organization of eukaryotic chromosomes o Complex organization needed to accommodate large size of genome o Associated with two major classes of proteins Histones Positively charged forms ionic bonds with negatively charged DNA H1, H2A, H2B, H3, H4 Play a major structural role in the formation of chromatin Non-histones Negatively charged Exist as large number of heterogeneous proteins Likely to perform roles in the regulation of expression of specific genes o DNA makes up about 1/3 of the mass of the chromosome o Successive level of DNA Packing Eukaryotic chromosomes contain an enormous amount of DNA To fit all 46 chromosomes into the nucleus, elaborate , multilevel system of DNA packing must take place DNA - 2nm wide - Double helix - Made up of 2 polynucleotide chains Nucleosomes Beads on a - Histones are responsible string - Positively charged histone bind to negativelycharged DNA - 4 types of histone, 2 of each type: H2A, H2B, H3 and H4 forms the core particle (histone octamer) - The DNA then coils around the octamer (146 DNA base pair wound 1.65 times around the protein core) - DNA coils + histone octamer core particle forms the nucleosome (beads) - The fifth histone, H1, forms the linker protein near the bead and helps to stabilize the 30nm fibre by neutralizing the negative charge on the DNA and by interacting with the histone octamer and nonhistone proteins - Nucleosomes can change shape and position to allow RNA-synthesizing polymerases to move 1|Page Created by SefLRho (2012) DHS

along DNA 30 nm chromatin fibre - With the help of H1, the beaded string coils with 68 nucleosomes per turn and forms a solenoid Looped domains 300nm - Chromatin fibres interact with chromosome scaffold made of non-histone proteins forming looped domains (700nm) Chromosome 1,400nm - The looped domains coil and fold - Compact itself into metaphase chromosome - The long arm of the chromosome is called q arm and the shorter arm is called p arm o Eukaryotic genome during interphase During interphase, chromatin exist in diffused state There are two types of chromatin: euchromatin and heterochromatin Most of the chromatin exists as euchromatin loosely coiled chromatin Generally transcriptionally active Parts of the euchromatin exists in 30nm fiber form, the more actively transcribed parts exists in the beads-on-a-string form and the very actively transcribed parts especially the promoter sequences within genes are depleted of nucleosomes to allow access t transcription factors easily Chromatin that are not actively transcribed are in the highly condensed stage of heterochromatin Located at the periphery of the nucleus Transcriptionally inactive The packing and unpacking of a chromosomal DNA provide a coarse adjustment for eukaryotic gene expression by making a region of DNA either more or less available for transcription. The fine-tuning beginning with the initiation of RNA synthesis-transcription. Describe the structure and organization of prokaryotic chromosomes o DNA in bacterial chromosome is associated with several types of DNA-binding proteins o Two called HU and H are small but abundant (positively charged) o Binds ionically to the negative charges of the phosphate group in DNA o Structurally similar to molecules called histones o But fewer, therefore chromosomes of prokaryotes can be considered as naked DNA o Include operon as part of structure* Compare the structure and organization of prokaryotic and eukaryotic chromosomes Prokaryotes Eukaryotes Both chromatin are coiled and lopped in a complex but orderly manner Associated with specific proteins Genome Size Small genome size Large genome size - Singular, circular - Multiple linear chromosome chromosomes - Might include - Found in membraneextrachromosomal bound nucleus plasmids - Found in nucleoid region Coding sequences Most of the DNA in prokaryotic Most of the DNA (98.5%) does genomes codes for proteins not code for any protins Non-coding sequences Only small amount of nonSome of the non-coding

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coding DNA consisting of mainly regulatory sequences such as promoters (no enhancers or silencers in prokaryotes) Few repetitive DNA, absence of introns

Compartmentalization

No. of chromosomes Origin of replication mRNA termination

Genes code for enzymes of related functions tend to be organized in clusters known as operons under the coordinated control of a single regulatory unit Form polycistronic mRNA DNA is transcribed into mRNA and used immediately in translation No compartmentalization of transcription and translation process during protein synthesis One circular molecule one per chromosome End with the terminator sequences

sequences are known to be regulatory sequences such as promoters, enhancers and silencers But most non-coding sequences have no known function. Presence of introns and repetitive DNA Each gene has its own promoter and are transcribed into monocistronic mRNA

Pre-mRNA has to be processed and introns have to be spliced out before the mRNA can be used for translation Compartmentalization in the cell Many linear molecules Many per chromosome End with poly A tail

Describe the structure and function of the non-coding regions of eukaryotic DNA with reference to intron, centromere, telomere, promoter, enhancer and silencer o Introns non-coding sequences found between exons in a molecule and is excised during mRNA processing serve a purpose in alternative RNA splicing alternative RNA splicing can produce different mature mRNA transcripts (one gene can now code for more than one polypeptides) introns enable alternative splicing and the subsequent production of multiple proteins from one gene o Regulatory sequences Promoters: nucleotide sequences located just upstream of the gene that attract and bind transcription factors and RNA polymerase to form the transcription initiation complex so that transcription can begin In eukaryotes, the promoter include the core promoter TATA box, CAAT box Similar in prokaryotes, initiation is also controlled by promoter sequences, TATAAT Pribnow box (-10 position) and also TTGACA (-35 position) o Repetitive DNA (Transposons and retrotransposons) Transposons are moderately repetitive sequences that are able to move about the genome They can be inserted into the middle of the coding sequences and interrupts the genes, within a sequence that is involved in the regulating transcription to

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increase or decrease the production of one or more proteins and just downstream of an active promoter to activate its gene Retrotransposons Move through an RNA intermediate and back into the DNA by reverse transcriptases Enhancers Sequences that binds to certain proteins (activators) that increase the rate of transcription by helping to form the transcription initiation complex Only present in eukaryotes Silencers Sequences that bind to certain proteins (repressors) that repress the rate of transcription Only present in eukaryotes Telomeres Region found at the end of the linear DNA of eukaryotic chromosomes (prokaryotes do not have telomere since most of their DNA is circular) Made up of non-coding regions, mostly tandem repeat sequences Each repeat is short (5-10 nucleotides) and the number of repeats range from hundreds to thousands of times, known as satellite DNA In human each repeat is made of guanine-rich, 6-8 nucleotides, typically in the form 5 TTAGGG 3 usually the telomere has a region at the 3 end which is single stranded known as 3 overhang this is a result of the end-replication problem due to the limitation of DNA polymerase to replicate all the way to the end of the chromosomes DNA polymerase can only add nucleotides to the 3 hydroxyl end of a pre-existing polynucleotide Thus there is no way to complete the 5 ends of the daughter DNA strands of the lagging strand Even if an Okazaki fragment can be started with an RNA primer bound to the very end of the template strand, once that primer is removed, it cannot be replaced with DNA as there is no 3 end onto which DNA polymerase can add DNA nucleotides Therefore, repeated rounds of replication thus produce shorter and shorter DNA molecules at the 5 end Centromere Most condensed and constricted part of the fully-condensed chromosome No defined DNA sequence Place where the two sister chromatids are joined together Cohesin proteins help to hold the two sister chromatids together Is also the site of assembly of protein kinetochore Region that may be the thinnest but it the strongest as it is subjected to the greatest tension when spindle fibres shorten DNA sequences of centromere are repeated in tandem not transcribed with considerable amount of heterochromatin vary greatly in length In humans, the primary centromeric repeat unit is called -satellite

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in this chromatin, the normal histone H3 is replaced with centromere-specific variant CENP-A in humans CENP-A is believed to be important for the assembly of the kinetochore on the centromere Each chromosome should have only 1 centromere, chromosomes that have two functional chromosomes will result in chromosomal breakage during mitosis. Describe the role of telomere o Role 1: protect the organisms genes from being eroded through successive rounds of DNA replication Postpones the erosion of genes near the ends of DNA molecules due to the endreplication problem Therefore genes will not be affected for the bulk of the organisms lifespan o Role 2: regulating replicative cell senescence The length of the telomere limit the no. of times the cell can divide This is known as hayflicks limit Cells with longer telomeres tend to go through a high number of cell divisions and survive longer than cells with shorter telomere Complete loss of telomere repeats eventually triggers cell death o Role 3: telomeres protect and stabilize the terminal ends of the chromosomes from exonuclease attack and the fusion of the ends of the linear chromosomes. Thus the integrity of a chromosomes structure and function is maintained telomeric sequences form special secondary structures with telomere-specific proteins they exist as a loop by tucking in the 3 single strand overhang into the same sequence in an upstream region of the telomere the 3 single stranded end will be able to complementary base pair with the upstream single-stranded region to form a T-loop the displaced original upstream telomere sequence will form the D-loop o Role 4: telomeres serve as template for telomerase activity, thus allowing its own extension, overcoming the built-in limit on the number of times cells can divide Telomeres do not shorten in every cell An enzyme called telomerase catalyses the lengthening of telomeres in eukaryotic germ cells, thus restoring their original length and compensating for the shortening that occurs during DNA replication Enzyme telomerase catalyses the lengthening of the telomeres in eukaryotic cells by reverse transcription This prevents chromosomal fraying and prevents the ends of the chromosomes from beigning processed as a double strand DNA break which could lead to choromosome-tochromosome telomere fusion Telomerase is a ribonucleoprotein complex that contains a short molecule of RNA (TERC: Telomerase RNA component) The RNA sequence found in the telomerase serve as the template for adding telomeric repeat sequences of TTAGGG by a specialized reverse transcriptase (TERT: telomere reverse transcriptase) to the 3 end of the telomeres The complementary strand of the telomere is then added by the usual combined actions of primase, DNA polymerase and ligases Telomerase is only present in germ cells and some rapidly dividing somatic cells eg stem cells and wbc. Its function usually reduced as an organism ages In cells that do not have telomerase, the DNA will get shorter and the cell would eventually die

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In prokaryotes, chromosomes are circular and thus do not suffer from replication termination Describe the role of centromere o Role 1: allow sister chromatids to adhere The heterochromatin state of the centromeres seem to be essential for the recruitment of cohesion complexes that hold the sister chromatids together o Role 2: allow kinetochore protein to assemble Kinetochore is important for the correct and successful segregation of sister chromatids during mitosis o Role 3: allow for pairing of homologous chromosomes Without a functioning centromere, improper chromosomal alignment and segregation will result, leading to aneuploidy Aberrations that lead to dicentric chromosomes (ie 2 centromere on 1 chromatid) may lead to chromosomal breakage during anaphase as the same sister chromatid is pulled in both direction to opposite poles of the cell Introducing of the control of gene expression o All cell types carry the same genetic material but differ dramatically in both structure and function o This is because not all proteins are produced in all cells or in the same amounts or at all times o Each cell of multicellular eukaryote only expresses a small fraction of its gene at a point of time, resulting in the synthesis and accumulation of different sets of RNA and protein molecules o Gene expression control mechanisms allow for differential gene expression o Cells can also respond to external signals bringing about a change to the expression of its gene through cell signaling o Gene regulation in eukaryotes take place at many levels but most commonly taking place at the level of gene transcription. This is because it saves resources and is easier to control Level of gene regulation Genetic code Transcriptional Post-transcriptional mRNA transport (not in syllabus) Mechanisms Control the number of copies of genes Control access to the genes, control when and how often a given gene is transcribed Control and regulate the splicing and processing of pre-mRNA after it has been produced Select which fully processer mRNA are retained in or exported out of the nucleus into the cytoplasm These mechanisms prevent translation These mechanism act after the protein has been produced such as selectively activating/deactivating, degrading etc

Translational Post-translational

Describe the process and significance of gene amplification within cells o Control at the level of genetic code involves Gene amplification DNA rearrangements (not in syllabus) o Gene amplication is a process that specifically increase the number of copies of the gene of interest which hence increases the overall rate of transcription and the amount of gene products o The process involves the repeated replication of the selected DNA sequence

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o o

This results in a cell which has a normal number of all other genes except for the gene of interest which exist in high copy number The significance of gene amplification is that More copies of the genes of interest will be available as templates for transcription => Gene expression is increased significantly => Leads to the production of multiple copies of RNA synthesis in the preparation for rapid growth of embryo Eg ribosomal RNA genes Amplified for the formation of large number of ribosomes for protein sy gene amplification can occue in several ways such as: repeated firing of DNA replication origins to increase the number of templates for transcription, yielding greatly increased protein and/or RNA product for genes at the amplified loci

unequal sister chromatid exchange incorrect pairing of two homologous chromosomes, resulting in unequal crossing-over during prophase I of meiosis, leading to duplication of the original gene

chromosome breakage existing across both sister chromatids, leading to two frayed ends which can fuse and result in a single chromosome with a duplicate set of genes due to unequal breakage

Slippage during DNA replication, followed by back-folding of the strand. Mispairing occurs between the daughter DNA strand and parental template strand causes parts of template DNA to be copied more than once

Define control elements and explain how control elements and other factors influence transcription o Control elements are segments of non-coding DNA that help regulate transcription by binding transcription factors o Control elements include Promoter Non-coding DNA immediately upstream of the gene Is the binding site for general transcription factors and RNA polymerase, forming a pre-initiation complex

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many promoters comprise of a TATA box which is important in determining the precise starting point for transcription as it is where TFIID binds. In eukaryotes, TATA box resides 20 to 30 bases (-20 to -30) upstream of the transcription start site In prokaryotes, the promoter consists of Pribnow-fragment (TATAAT) which is similar to the TATA box. The pribnow-fragment is located at position -10 Eukaryote promoters also comprise a CAAT box at 50-130 bases upstream Proximal control elements Non-coding DNA that usually lies directly upstream of the promoter Bind to certain specific transcription factors Distal control elements Non-coding DNA that is located further away from the gene of interest than proximal control elements Can be found thousands of nucleotides upstream or downstream of the gene, sometimes even within an intron Bind to certain specific transcription factors Enhancer Region containing a group of distal control elements Activators binds here to increase the rate of transcription - Bound activator proteins interact with components of the transcription machinery which includes general transcription factors and RNA polymerase. - This results in the improved recruitment of general transcription factors and RNA polymerase to form a transcription initiation complex. - This also facilitates the proper positioning of the transcription initiation complex on the promoter to initiate transcription There may be multiple enhancers for a particular gene Some genes may not have enhancer Only found in eukaryotes Silencers Region containing a group of distal control elements Non-coding DNA located further away from the gene Repressors bind here to decrease the rate of transcription - This is because the binding of DNA-binding proteins required to initiate transcription is blocked - Assembly of transcription initiations is prevented and transcription is blocked There may be multiple silencers for a particular gene o General transcription factors + RNA polymerase II + enhancers + specific transcription factor = active transcription initiation complex State the various ways in which gene expression may be controlled at transcriptional level o Change in conformation of the DNA region of interest As DNA can be packaged into chromatin and unfolded chromatin has beads-on-a-string appearance, gene expression can be controlled by modifying/remodeling of the structure of chromatin to alter the accessibility of DNA to regulatory proteins Tightly packed chromatin is made more accessible for transcription by changing its packing structure to a more open conformation (eg conversion of heterochromatin to euchromatin) Two important processes to do with this are 8|Page Created by SefLRho (2012) DHS

DNA methylation Histone modification DNA methylation Methyl groups are added to cytosine nucleotide in the sequence 5-CG-3 (CpG dinucleotides) by enzyme DNA methyltransferace Usually occurs at the promoter regions Prevents the binding of transcriptional proteins required for transcription initiation and increase in the condensation of chromatin through the recruitment and action of histone deacetylases (HDACs) Makes genes less accessible Silences the gene De-methylation will turn inactive genes on. Histone modification N-terminus of each histone molecule protrudes outwards forming histone tails Histone tails are rich in lysine residues and are positively-charged which interact favorably with negative-charged phosphate group of DNA, increasing the affinity of DNA to ribosome surface Various enzymes can modify these histone tails and affect the accessibility of DNA for transcription initiation Acetylation - Lysine residues in histeon tails are acetylated by enzyme histone acetyltransferace (HAT) - Lysine molecules becomes uncharged, resulting in the reduction of the affinity of histone complex to DNA - As histone is less tightly bound to DNA, DNA strands become less condensed and more open, making DNA regions exposed, activating transcription - The acetylated lysine can also serve as binding sites for certain transcription factors Deacetylation - Enzyme histone deacteylase (HDAC) catalyses the deacetylation of acetylated lysine residues in histone tails - Lysine becomes positively charged again, increasing affinity for histone to DNA - Prevents transcription Control elements and transcription factors Transcription in eukaryotes requires various regulatory proteins called transcription factors. These factors help RNA polymerase II to bind. Therefore, by controlling these transcription factors, we can control the gene expression Control elements are involved in the initiation of transcription, together with transcription factors The transcription in eukaryotes is much more complex than prokaryotes (use of operon, promoter and regulators) because eukaryotes have more complicated cell structure that include many proteins and a variety of cell organelles most eukaryotes are multicellular and have many different cell types, making cell differentiation important to generate these cell types Eukaryotes progress through developmental stages that require changes in gene expression. By switching genes off and on, cells can prevent resources from

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wastage. A typical human cell normally expresses about 3-5% of its genes at any given time or 20% also accepted DNA structure of eukaryotes in much more complex than that in prokaryotes, thus protein-DNA interactions are also more complex Eukaryotic transcription and translation are spatially and temporally separate, unlike that in prokaryotes. The recruitment of enzymes and transcription-related proteins is thus more complex Transcription factors Transcription factors are DNA-binding proteins that bind to the control elements of gene together with the RNA polymerase It is generally accepted that cis-acting regulatory sites influenced transcription initiation by acting a binding sides to transcription regulatory proteins Only when the complete transcription initiation complex has assembled can the RNA polymerase begin to move along the DNA template strand, producing a complementary strand of RNA There are 2 types of transcription: - General transcription factors - Specific transcription factors General transcription factors - essential for the transcription of all protein-coding genes as they are required to assemble the initiation complex - bind to the promoter which is located just before the gene - are needed before RNA polymerase II can bind to the promoter - some bind to the DNA sequence independently such as TFIID which binds to the TATA box 5-TATAAAA-3 while others primarily bind to proteins such as each other and RNA polymerase II - interaction of general transcription factors and RNA polymerase II with promoter leads to low basal rate of initiation Without general transcription factor, RNA polymerase cannot bind effectively because of low affinity 5-10% With general transcription factor, affinity between RNA polymerase II and promoter increase ~30-50% (from 50% to 100% depends on specific transcription factors Specific transcription factors - Recognize and bind to control elements (enhancers/silencers) beside the promoter, which are located relatively further away from the gene

Known as activators/repressors when they stimulate higher/lower levels of transcription of certain genes Promote the assembly of pre-initiation complex (general transcription factor and RNA polymerase II) Determine the ultimate level of transcription These transcription factors need to bind to proteins at the promoter before they can increase transcription rates

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Despite specific transcription factors being located much further away from the promoter region, these transcription factors can cause DNA to bend, forming a loop and bring themselves into close proximity to the RNA polymerase o Combinatorial control of gene expression Particular combination of control elements and transcription factors regulates the expression of any given gene (not just have and dont have but have HOW MUCH and what TYPE YOU have) Affected by the type and number of control elements and transcription factors Certain combinations have different impact and influence on the gene expression and can generate a great deal of cell types from relatively few gene proteins o Coordinately controlled genes Genes of related function often need to be turned on/off together In prokaryotes, this is done by operons regulated by singer promoter In eukaryotes, genes of related function are often scattered all over the chromosomes and when these genes need to be expressed together, simultaneous transcription of the genes is usually done in response to external chemical signals These chemical signals stimulates the transcription of all the genes that recognize this signals allowing for simultaneous expression of related genes Thus the location of each gene would no longer matter Describe the eukaryotic processing of pre-mRNA in terms of intron splicing, polyadenylation and 5 capping o RNA processing is necessary in eukaryotes to modify pre-mRNA to mature mRNA for translation o Involves 5 7-methylguanosine capping, addition of 100-250 adenine residues for form poly A tail, splicing of introns and exons + editing of mature mRNA sequences o Do not occur in prokaryotes because transcription and translation in pro are simultaneous o 7-methylguanosine 5-capping Linked via a 5 -> 5 triphosphate bridge to the first nucleotide Added first, co-transcriptionally Improves the stability of the pre-mRNA chain by preventing it from degradation Important for recognition of the mature mRNA by ribosomes to allow for translation initiation o Polyadenylation at 3 end Addition of adenine nucleotides Linked via covalent phosphodiester bond Added by polyadenylate polymerase No template is needed Protects mature mRNA from degradation by RNases Help in the export of mNA The length of poly-A tail determines the lifespan of the mRNA Once in the cytoplasm, poly A tail is continuously shortened by enzymes Once it becomes too short, it triggers action of enzymes to remove 5 cap and degradation of RNA Indirectly, length of A tail affect the number of copies of polypeptides that can be produced before mRNA is degraded o Splicing Introns are excised while exons are spliced together Carried out by spliceosome

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Large complex comprising of sub-units called snRNPs (small nuclear ribonucleoproteins) spliceosome cut at splice sites which are determined by the sequences of nucleotides at intron-exon boundaries requires energy ATP variations in splicing produces different mRNA and increases the size of the genome o Editing of mature mRNA sequence Nucleotide sequences are modified Maybe edited to add new nucleotides to form different polypeptides from the same genetic code o Mature mRNA has coding regions and untranslated regions (UTRs) State the various ways in which gene expression may be controlled at translation level (RNAInterference) o Half-life of mRNA Prokaryotic mRNA have very short lives and are degraded by enzymes after only a few minutes In eukaryotes, mRNA have different half-lives depending on features such as 5-cap and length of poly A tail In addition, 5 and 3 UTRs also affect the stability of mRNA by influencing its affinity for RNases The enzymatic shortening of the poly A tail and the removal of the 5 cap causes nuclease enzymes to rapidly destroy the mRNA o Initiation of translation Translation initiation factors are proteins and are required in processes such as scanning the mRNA for start codon, locating the binding sit of initiator tRNA to the AUG start site and formation of the initiation complex By varying the among of translation initiation factors, rate of translation initiation is controlled Phosphorylation and dephosphorylation of one of more of the initiations factors Phorphorylation activates the translation initiation complex while dephosphorylation deactivates the initiation complex Regulatory proteins that can block the initiation of translation RNA silencing mechanisms Small RNAs can interfere with the expression of a mature mRNA Micro-RNAs (miRNAs) - Short single-stranded mRNA - 20-25 nucleotides long - Derived from longer precursor mRNA, unmatched segment of RNA precursor, and from hairpin structure - RNA precursors cleaved by enzyme Dicer resulting in miRNAs pair with UTRs of other mRNA - Blocks translation or target mRNA for degradation siRNAs - synthesized from double-stranded segments of matched mRNA - enzyme Dicer cuts the double-stranded RNA molecule into short fragments - sense strand is degraded while antisense strand associate with RNAinducing silencing complex (RISC)

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siRNA + RISC bind to any mRNA molecule that have complementary sequence and blocks translation/mark it for degradation mRNA transport (not in syllabus) transport of mRNA through the nuclear pores regulated State the various ways in which gene expression may be controlled at post-translation level o Polypeptides are usually modified before they are functional o Biochemical modification Phosphorylation is a common mechanism for controlling protein behavior Adding functional groups can activate/deactivate proteins o Protein degradation Regulates how long a protein remains and functions in the cell Attaching ubiquitin commonly marks the proteins for destruction Ubiquitin-protein complex is recognized by proteosome which unfolds the proteins and destroys it within a central cavity Proteins are digested into small peptides and amino acids Outline the differences between prokaryotic control of gene expression within the eukaryotic model Prokaryotes Eukaryotes Gene expression only at transcriptional and Transcriptional, post-transcriptional, post-translational level translational and post-translational No control at genetic code level Gene amplification + DNA arrangements No control at chromosomal structure level DNA methylations + histone modification Simple transcriptional initiation complex Complex transcriptional initiations complex - Sigma factor - General TFs recognize promoter before RNA can bind - Presence of specific transcription factors and other proteins eg DNA bending proteins No distal control elements Distal control elements eg enhances, silencers No DNA bending DNA bending to bring activators in close proximity to the RNA polymerase and general TFs No combinatorial control Combinatorial control of control elements and specific TFs Polycistronic mRNA Monocistronic mRNA Simultaneous expression of a few genes done Simultaneous expression of a few genes done via operons via coordinately controlled mechanisms Triggering of related pathways via a common signal Up-regulation of transcription done by coUp-regulation done by activators activators eg cAMP and increasing affinity of RNA polymerase Down-regulation of transcription done by Down-regulation of transcription done by decreasing affinity of RNA polymerase specific TFs and repressors No processing of mRNA Post-transcriptional processing of mRNA mRNA degradations occur within minutes. mRNA more stable and duration of life Duration controlled by poly A tail. controlled by length of poly A tail. Long => more stable Anti-sense RNA from non-coding DNA can bind Production of small mRNA cut by Dicer to to complementary mRNA and inhibit degrade mature mRNA with complementary

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translation

sequences to the small mRNA Similarities Presence of proximal control elements. Pribnow box vs TATA box. Phosphorylation of certain initiation factors to control initiation Biochemical modification of proteins present in both. Phosphorylation of polypeptide chains/addition of functional groups to enhance/repress effect of proteins

Describe the functions of common proto-oncogenes and tumour suppressors gene and explain how loss of function mutation and gain of function mutation can contribute to cancer o Proto-oncogenes found in normal human cells carrying codes for growth stimulatory proteins able to encourage cells to multiply can be turned on/off undergo gain of function mutation to form oncogenes which continuously stimulate the development of cancer by instructing cells to make proteins that stimulate excessive cell growth Translocation: chromosomes break and rejoin incorrectly, translocating fragments from one chromosome to another and a proto-oncogene may end up near an especially active promoter which increases the transcription of the gene, making it oncogene Viral integration: insertion of a retrovirus genome may activate it eg some retroviruses carry oncogenes in their genome Gene amplification: increases the number of copies of gene Point mutations: occur spontaneously or from environmental influences which may change the gene to be hyperactive or change sequences of promoter/enhance to increase rate of transcription Eg of proto-oncogene: ras gene Involved in cellular signal transduction Activation of ras gene causes cell growth, differentiation and survival RAS can be switched on/off by the binding of either guanosine disphosphate or guanosine triphosphate Mutation can occur and cause RAS to be locked in active stage by preventing the hydrolysis of guanosine triphosphate to disphosphate Active RAS continuously transfer growth promoting signals and constantly stimulate the cell to divide o Tumor suppressing gene Stop the cell from multiplying These genes are normally present in the cell to prevent cell from accumulating DNA damage by repairing damaged DNA and inhibiting the cell cycle Some can control the adhesion of cells to each other to stop cell from dividing due to adhesion pressure Absence/damage can lead to non-stop cell division even if DNA is damaged Results in loss of function mutation Eg p53 Promotes synthesis of cell cycle-inhibiting proteins Usually synthesized but rapidly degraded or inactivated by other proteins Only activated by DNA damage or cell damage It can activate DNA repair genes but if repair is impossible, it activates cell death

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However when mutated, p53 is locked in inactive state and cells proliferate rapidly Eg Retinoblastoma gene Most tumor repressor genes are recessive mutations ie both alleles need to be mutated in order for function to be ceased Describe the development of cancer as multi-step process o Cancer is a large class of diverse diseases which causes uncontrolled cell growth and division o All cancers first involve the accumulation of genetic changes o Mechanisms that can lead to such mutations include translocation, transposition, viral integration, gene amplification and point mutations o May develop by clonal evolution Single cell genetically mutated One of the descendent cells mutate further Cells that become cancerous produces daughter cells that also inherit the mutations and are cancerous o Effect: cancer cells lose control over cell proliferation Do not respond to growth inhibition signals or continuously send growth signals to nucleus even without external growth signals Unable to enter G0 phase Does not respond to contact inhibition A benign tumor is formed o Progress of normal cell to cancer cell is multi-step involving gain-of-function mutations and lossof-function mutations o At least 5-6 mutations must take place for cell to become fully cancerous o Usually it is the mutation of 1 gain-of-function mutation in proto-oncogene eg ras gene and several loss-of-function mutation in tumor suppressor genes eg p53 This is because tumor suppressor genes are often recessive mutations while most oncogenes behave as dominant allele requiring only 1 mutation o Malignant cancer cells can metastasize and invade other tissues by blood stream / lymph vessels resulting in secondary cancer o Malignant tumor is able to produce signals that cause the development of blood vessels towards itself to supply nutrients and oxygen

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