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Chapter 7

BIOL 205 Chapter 7: BIOL205 Chapter 7 Textbook Notes

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BIOL 205
Ian Chin- Sang

BIOL205 NOTES 7.1 DNA: The Genetic Model 1. Genes were known to be associated with specific traits, but physical nature was not understood. Mutations were known to alter gene function, but chemical nature was unknown. 2. One-gene-one-polypeptide hypothesis postulated that genes determine the structure of proteins and other polypeptides. 3. Genes were known to be carried on chromosomes. 4. The chromosomes were found to consist of DNA and protein. 5. The results of a series of experiments beginning in the 1920s revealed that DNA is the genetic material. The experiments described next showed that bacterial cells that express one phenotype can be transformed into cells that express a different phenotype and that the transforming agent is DNA. Discovery of Transformation - Bacterium Streptococcus pneumoniae is lethal in mice, but some strains are less virulent - Griffith used two strains that are distinguishable by colony appearance - R strain, the non-virulent one, was rough in appearance with no polysaccharide coat - S strain, the fatal one, has a polysaccharide coat - Griffith heated the virulent ones, injected them into mice, and they did not die: suggests that the carcasses of cells do not lead to death o Mice with heat-killed and non-virulent did die and live cells could even be recovered from them - The cell debris of boiled S strain had converted the R strain into live S cells: called transformation - After many tests, they found that only when enzyme DNase, which breaks up DNA, was inserted to destroy the genetic material, did the transforming stop o This further suggests that DNA is the transforming agent Hershey-Chase Experiment - Used T2 phage, a virus that infects bacteria o Most of structure is protein, with DNA contained inside the protein head o Used radioisotopes to track each kind of material in the process o Phosphorus is not found in AA building blocks of proteins, but integral in DNA o Integrated P into phage DNA and S into proteins of another phage 1 BIOL205 NOTES - Infected 2 E. coli cultures, one with phosphorus and the other with sulfur radioisotope - After time, sheared empty phage carcasses (ghosts) from bacterial cells, separating in centrifuge o Measured radioactivity in 2 fractions o 32P-labelled phages: radioactivity ended up inside bacterial cells (phage DNA entered the cells) o 35S-labelled phages: radioactivity ended up in ghosts, so did not enter - Conclusion: DNA is the hereditary material 7.2 DNA Structure Three key properties: 1. Faithful replication of genetic material at every cell division is crucial because we have virtually the same genetic material in every cell. Must allow faithful replication. 2. Must encode constellation of proteins expressed by an organism and so, genetic material must have informational content. 3. Because mutations provide material for evolutionary selection, genetic material must be able to change but structure must remain stable. DNA Structure before Watson and Crick - The building blocks of DNA: DNA contains three chemical components (1) phosphate, (2) a sugar called deoxyribose and (3) four nitrogenous bases – adenine, guanine, thymine and cytosine o Has an H atom at 2’-carbon atom whereas RNA has an OH group o Latter, pyrimidines, have single ring structure and others, purines, have double ring structure o Nucleotides are made up of phosphate group, sugar molecule and a base - Chargaff’s rules of base composition o Total pyrimidines (C + T) equal total purine nucleotides (A + G) o Amount of A equals amount of T & amount of C equals amount of G ▪ These two do not have to equal each other in proportion 2 BIOL205 NOTES - X-ray diffraction analysis of DNA: X rays fired at DNA fibres and scatter of rays is observed by catching on photographic film o Angle of scatter by each spot gives information about position of an atom in the DNA molecule o Suggested that DNA is long and skinny and has two similar parts that are parallel to each other in a spiral-like fashion o Allowed them to deduce 3D structure of DNA The Double Helix - 3D structure composed of two side chains of nucleotides twisted into a double helix o Held together by hydrogen bonding between bases of each strand o Backbone made of phosphodiester linkages o Phosphodiester linkage connects 5’-carbon atom of one deoxyribose to 3’- carbon atom of adjacent deoxyribose - Backbones are antiparallel in orientation - Each base attached to 1’-carbon atom of deoxyribose sugar in backbone of each strand; faces inward toward base on other strand - Base pairs stack on top of each other in middle of double helix, adding to the stability by excluding water molecules o Two distinct sizes of grooves running in a spiral: major grooves and minor grooves; most are major grooves - Purine always pairs with a pyrimidine base o Make note that C-G has three hydrogen bonds whereas, A-T only has two o DNA with more C-G pairs is more stable than with many A-T bonds o When exposed to heat, the former require higher temperatures to be denatured - Fulfilled the three requirements for a hereditary substance 1. Some sort of genetic code may write information in DNA as a sequence of nucleotides and then translate into AA sequences in proteins 2. Mutation is possible by substitution of one type of base for another at one or more positions 3. Double helix allows for “copying mechanism for genetic material” 3 BIOL205 NOTES 7.3 Semiconservative Replication - Think of it as an unwinding of the two strands that will expose a single base on each strand that has ability to bond with complimentary nucleotides in solution o Each of two singles strand acts as a template strand to direct the assembly of complimentary bases to re-form double helix identical to original - Each daughter molecule should contain one parental nucleotide chain and one newly synthesized nucleotide chain - Semiconservative replication is where the double helix of each daughter DNA contains one newly synthesized strand and one from original - Conservative is where parent DNA molecule is conserved and a single daughter double helix is produced consisting of two newly synthesized strands - Dispersive is where daughter molecules consist of strands each containing segments of both parental DNA and newly synthesized DNA Meselson-Stahl Experiment - Set out to discover which form of replication DNA followed - Grew E. coli cells in a medium containing heavy isotope N and was inserted into the nitrogen bases which are then set to be incorporated into DNA strands o After many divisions, they were labelled with this heavy one o Cells were removed from 15 and put in normal 14 and samples were taken - They were able to distinguish DNA of different densities due to cesium chloride gradient centrifugation o Cesium and chloride ions tend to be pushed to bottom of tube during centrifugation o Gradient of ions is created in the tube and bands are formed at different densities - Shown that DNA is intermediate density shown half blue N and half gold N4 - After two generations, both intermediate and low-density were shown, confirming the semiconservative model 4 BIOL205 NOTES The Replication Fork - Replication zipper is found in DNA molecule during replication and is the site where double strand will unwind to produce two strands o Serve as templates for copying o Used H – thymine nucleotide labelled with a radioactive hydrogen isotope called tritium o Each newly synthesized daughter molecule should then contain one radioactive “hot” strand with H and another one without – “cold” - After many replications in “hot” medium, bacteria was lysed and cell contents settled onto grid for electron microscopy, covered with emulsion and exposed to dark for 2 months o As H decays it produces beta particle, it will emit beta particle onto emulsion o Ring of dots appeared on autoradiograph o Thick curve of dots cutting through interior of circle was newly synthesized strand, with two radioactive strands DNA Polymerases - How are bases brought to double helix template - E. coli bacteria was used and DNA polymerase was isolated o Enzyme adds to 3’ end of growing chain using exposed DNA strand as template - Substrates for polyemerase are dATP, dGTP, dCTP and dTTP o With each new base added, 2 of 3 phosphates is removed to form pyrophosphate (PP) i o Energy produced by cleaving this high energy bond and hydrolysis of pyrophosphate helps drive DNA polymer synthesis 5 BIOL205 NOTES 7.4 Overview of DNA Replication - As DNA pol III moves forward, double helix is continually unwinding ahead of enzyme to expose further lengths of single DNA strands that will act as template o Acts at the replication fork but because polymerase adds to 3’ growing tip, only one of two antiparallel strands can serve as template ▪ Called the leading strand: synthesized in a smooth manner - Synthesis on other template takes place on 3’ as well, but in wrong direction o Cannot go on for long o Must be in short segments called Okazaki fragments that polymerase synthesizes in a segment, moves back to 5’ end where growing fork has exposed new template and begins process again - Synthesis of both strands must be initiated by a primer, or short chain of nucleotides, that binds to template strand o Primers are synthesized by primosome, including primase, a type of RNA polymerase o On leading strand, only one primer is needed o On lagging strand, every fragment needs its own primer - Pol I removes the RNA primers with 5’ to 3’ activity - DNA ligase joins 3’ end of gap-filling DNA to 5’ end of downstream Okazaki (lagging) o Joins broken pieces of DNA by catalyzing formation of a phosphodiester bond between 5’-phosphate end of one and adjacent 3’ of another - Mismatched BP occurs when 5’-3’ polymerase activity inserts a wrong nucleotide and this is often due to tautomerization o Tautomers are isomers where positions of their atoms and bonds between - Fortunately, mismatches are usually removed by 3’-5’- exonuclease activity o After it is removed, polymerase has another chance to add correct base o Those lacking exonuclease, will have more mutation - RNA primer is more likely than DNA to contain errors because primase lacks a proofreading function o Only after RNA primer is gone does DNA pol I catalyze DNA synthesis to replace the primer 6 BIOL205 NOTES 7.5 The Replisome: A Remarkable Replication Machine - Speed is another hallmark of DNA replication o 5 million BPs can be copied at a rate of 2000 nucleotides per second o We know that E. coli uses only 2 strands and so, each one must do 1000 nucleotides per second - DNA polymerase is part of a complex called replisome - DNA pol III is part of pol III holoenzyme that has two catalytic cores and many accessory proteins o One core handles synthesis of leading strand while other is lagging o Some accessory proteins form a connection between the two cores o Important accessory is the beta clamp that encircles DNA like a donut ad keeps pol III attached to molecule ▪ Pol III transformed from distributive enzyme, that can only add 10 nucleotides before falling off, to processive enzyme, that can add tens of thousands - Note that primase is not touching the clamp protein, so acts as distributive enzyme o Only needs to form starting complex for DNA pol III Unwinding the Double Helix - Replisome contains two classes of proteins that open the helix and prevent over-winding: helicases and topoisomerases, respectively o Helicases disrupt hydrogen bonds that hold two strands of double helix together ▪ Fits like a donut around the DNA and rapidly unzips ahead of synthesis ▪ Unwound DNA stabilized by single-strand- binding proteins that prevent duplex from re-forming - DNA gyrase can treat supercoiling by relaxing the extra twisting that is created when the replication fork opens up o Relax by breaking either a single DNA strand or both, allowing DNA to rotate into a relaxed molecule o Topoisomerases finish by rejoining strands Assembling the Replisome: Replication Initiation - Assembly beings a precise locations called origins and only at certain times in life o In E. coli, begins in locus (oriC) and then goes to both directions until forks merge - DnaA binds to specific 13 bp sequence called DnaA box, repeated 5 times in oriC o Origin is then unwound cluster of A and T (only held by 2 H bonds) - After unwinding begins, more DnaA proteins bind to newly unwound SS regions - Two helicases (DnaB) now bind and slide in 5’-3’ direction to unzip - Primase and DNA pol III holoenzyme are now recruited to replication fork for synthesis 7 BIOL205 NOTES 7.6 Replication in Eukaryotic Organisms - Very similar to that of prokaryotic because both use semiconservative model - Number of replisome components also increases Eukaryotic Origins of Replication - Bacteria like E. coli usually complete replication in 20-40 minutes, but in eukaryotes, it can take yeast 1.4 hours and cultured animal cells 24 hours o Can last up to 100-200 hours in some cells - Yeast were used to identify eukaryotic proteins because of their simplicity o Similar to oriC in E. coli - 100-200 bp origins have conserved DNA sequence that includes an AT-rich region that will also melt when an initiator protein binds to adjacent sites o Each eukaryotic cell has many replication origins to replicate the larger genomes o Thousands of replication forks in 23 human chromosomes o Proceeds in both directions from multiple origins o Double helices made at each origin will elongate and eventually meet - This process results in two identical daughter molecules of DNA DNA Replication and the Yeast Cell Cycle - Synthesis of DNA takes place in the S phase of eukaryotic cell cycle - In yeast, 3 proteins are required to begin assembly of the replisome - Origin recognition complex (ORC) binds to sequences in yeast origins, much like DnaA o This process recruits two other proteins, Cdc6 and Cdt1 o All of these then recruit helicase (MCM complex) and other components of replisome - Cdc6 and Cdt1 are created in the G1 phase and are destroyed after synthesis has begun o Replisome can only be formed before S phase and none after this 8 BIOL205 NOTES Replication Origins in Higher Eukaryotes - Origins of replication in higher eukaryotes are much longer than other eukaryotes and have limited sequence similarity - In higher eukaryotes, unlike yeast, ORCs probably don’t recognize specific sequences o Much harder to isolate origins of humans because cannot use isolated DNA sequence to search - They do not interact with specific sequence scattered throughout the chromosomes, but interact indirectly with origins by associating with other proteins bound to chromosome o Gene rich areas replicate early in S phase and opposite in late o ORCs may, for example, have higher affinity for origins in open chromatin and bind to these origins first, THEN after this is replicated, bind to condensed 7.7 Telomeres and Telomerase: Replication Termination - Replication process replicates most of the chromosomal DNA, but there’s a problem with replicating the two ends, called telomeres o For leading strand, can go right up to this end o Lagging strand requires primers ahead of this, so when the last one is removed, sequences are missing at that end of the strand ▪ Single stranded tip remains in one of daughter DNA molecules ▪ If this daughter strand was replicated again, it would become a shortened double-stranded molecule after replication - Cells have evolved to prevent this loss o Addition of multiple copies of a noncoding sequence to the DNA at chromosome tips and so only these are lost, that contain no information - Essentially ends of chromosomes made up sequences repeated in tandem o Human chromosomes end in about 10-15 kb of tandem repeats of TTAGGG - Telomerase is an enzyme that adds the short repeats to the 3’ ends of DNA o Also carries a small RNA molecule that can act as a template for the telomeric repeat unit Process shown in figure: - Telomerase RNA molecule first anneals to the 3’ DNA overhang - Then extended with telomerase’s two components - After addition of a few nucleotides to the 3’ overhang, telomerase RNA moves along the DNA so that it can be further extended - 3’ end continues to be extended - Primase and DNA polymerase use very long 3’ overhang as a template to fill in the end of the other DNA strand 9 BIOL205 NOTES - RNA is serving as the template for the synthesis of DNA, whereas normally it’s the other way around - Telomeres associate with proteins to form protective caps that sequester the 3’ single- stranded overhang o Without this, double stranded ends of chromosomes would be mistaken for double stranded breaks by cell ▪ These are normally dangerous and lead to instability - Somatic cells produce very little telomerase so chromosomes or proliferating somatic cells get progressively shorter with each cell division until cell stops all divisions o Telomerase shortening and aging o Werner syndrome experience early onset of many age-related events including wrinkling of the skin, cataracts, graying of the hair etc. ▪ These people have shorter telomeres – mutation in WRN gene that encodes helicase that associates with proteins that comprise the telomere cap ▪ Chromosomal instability and premature-aging phenotype 10 BIOL205 NOTES - Genes of higher eukaryote are usually composed of exons that are coding, expressed regions, and introns, non-coding regions - A spliceosome removes the introns and joins the exons in a process called RNA splicing - Humans have about 21 000 genes that encode for 100 000 proteins because of alternative splicing - Non-protein-coding RNA (ncRNA) perform many essential roles - RNA copy of the gene must be synthesized in a process called transcription o First step is to copy, or transcribe, the information into a strand of RNA, using DNA as a template o Converted into an amino acid chain (protein) by translation - In eukaryotes, these take place in two different places, the former being in the nucleus and the latter being in the cytoplasm o Before going into the cytoplasm, undergoes removal of introns, 5’ cap and Poly-A tail addition - Two principles that this is based on 1. Complementarity of bases is responsible for determining sequence of RNA transcript in transcription 2. Certain proteins recognize particular base sequences in DNA and RNA – binding proteins bind to here and act on them 8.1 RNA Early Experiments Suggest and RNA Intermediate - Volkin and Astrachan found that when they infected E. coli with a T2 phage, a quick burst of RNA synthesis occurred, but “turned over” quickly o Brief lifetime, but suggested that it may be necessary in expression of T2 genome to make more virus particles - Pulse-chase experiment: infected bacteria are fed radioactive uracil (needed for only RNA synthesis so that it is labelled) and then after a while, rinsed and fed non- radioactive uracil o This “chases” the label out of RNA because radioactive ones break down and only the other ones are available o RNA analyzed shortly after “pulse” of radioactivity, is labelled, and other is not Properties of RNA 1. Ribose sugar in nucleotides rather than deoxyribose; RNA contains a hydroxyl group (OH) on the 2’ carbon atom and DNA only has an H 2. RNA is usually single-stranded nucleotide chain, not double stranded helix a. Much more flexible -> Intramolecular base pairing (with its own bases) 3. RNA molecules contain the pyrimidine base uracil (U) instead of thymine (T) a. Forms two hydrogen bonds with adenine just like T b. Capable of pairing with G (only during RNA folding, not transcription) c. U and G is weaker than U and A, but this versatility allows for complicated structures to be composed 4. RNA can catalyze biological reactions with ribozyme (RNA protein enzymes) 11 BIOL205 NOTES Classes of RNA - Messenger RNA (mRNA): encodes information necessary to make proteins o Gene influences phenotype through gene expression o Only an immediate intermediate product - Functional RNA: does not encode information to make protein, but is the final functional product o Never translated into polypeptides** o Transfer of information from DNA to protein, processing of RNA, regulation of RNA and protein levels in the cell o Transfer RNA (tRNA) are responsible for bringing correct AA to the mRNA during translation o Ribosomal RNA (rRNA) major components of ribosomes that guide assembly of AA chain by mRNAs and tRNAs - Encoded by a small number of genes, but rRNAs account for a very large % of RNA in cell - Functional RNA Specific to Eukaryotes: o Small nuclear RNAs (snRNAs) further process RNA transcripts; some unite with several protein subunits to form spliceosome o MicroRNAs regulate the amount of protein produced by many eukaryotic genes o Small interfering RNAs (siRNAs) and piwi-interacting RNAs protect integrity of plant and animal genomes - Long noncoding RNAs (ncRNAs) transcribed from most regions of the genomes of humans and other animals and plants; function unknown - These RNAs are constitutive, so continually synthesized 8.2 Transcription - RNA produced by process that copies the nucleotide sequence of DNA: transcription o RNA is then called a transcript Overview: DNA as Transcription Template - Two strands of DNA separate and one acts as the template for RNA synthesis - In any one gene, only one strand is used and in that gene, it is always the same strand, starting at the 3’ end of template gene - Nucleotides form stable pairs with their complimentary bases in template o Placed by RNA polymerase linking ribonucleotides and making an ever-growing strand of RNA - RNA grows in 5’ to 3’ direction (always added to the 3’ end) o Because nucleotides are oppositely oriented, this means that the template strand must be organized from 3’ to 5’ - Unwinds DNA ahead of time and rewinds the DNA that has already been transcribed - Nucleotide sequence of RNA is the same as the nontemplate strand of DNA, except T’s are replaced with U’s o Nontemplate of DNA = coding strand 12 BIOL205 NOTES Stages of Transcription - Because DNA is a continuous sequence, transcriptional machinery must be directed to the start of a gene and continue down the length until the end - Three distinct stages: 1. Initiation in Prokaryotes ▪ RNA polymerase binds to DNA promotor close to start of transcribed region – also called the 5’ regulatory region ▪ Non template is usually shown because they are in the same orientation ▪ Promotor is upstream of initiation site (5’ end of gene; downstream is later in the transcription process ▪ Upstream nucleotides are indicated by (-) and downstream by (+) ▪ First DNA base to be transcribed is +1 13 BIOL205 NOTES This figure shows an E. coli gene and 7 different promotors: -35 and -10 regions have great similarities (35 and 10 base pairs upstream of first transcriptional base) - Do not have to be identical to arrive at same set of nucleotides: consensus sequence - RNA polymerase holoenzyme binds to DNA here, unwinds, and begins RNA synthesis - Intervening part is 5’ untranslated region (5’ UTR) ------------------------------------------------------------------------------------------------------------------------------- ▪ RNA polymerase holoenzyme scans the DNA for a promotor region • Two alpha subunits, one beta, one beta’ and one w • Also includes sigma factor ▪ Alpha: assemble enzyme; promote interaction with regulatory proteins ▪ Beta: active in catalysis ▪ Beta’: binds to DNA ▪ W: enzyme assembly and gene expression ▪ Sigma: binds to -10 and-35 regions; separates DNA strands around -10; dissociates from rest of complex after initiation • Other sigma factors can recognize different promotor sequences 2. Elongation in Prokaryotes ▪ RNA polymerase unwinds DNA ▪ Region of single stranded DNA is called transcription bubble where template strand is exposed and polymerase monitors addition of ribonucleoside triphosphate to DNA template if it matches • Energy needed for this is derived from splitting high-energy triphosphate ▪ Rewinds DNA that has already been transcribed 14 BIOL205 NOTES 3. Termination in Prokaryotes ▪ Continues past protein-encoding segment of the gene, creating a 3’ untranslated region (3’ UTR) at the end of transcript ▪ Continues until termination signal reached ▪ Two major mechanisms for termination of E. coli • Intrinsic is direct: terminator sequences are 40bp, ending in GC rich region followed by strong of 6 or more A’s o Because this will result in CG rich area in RNA, the bases can bond with each other (internally) forming a hairpin loop – GC stems are more stable than AU o Loop followed by string of U’s o Strength of hybrid (DNA-RNA) in bubble is determined by amount of G-C pairs compared to A-U; will backtrack o Polymerase pauses after U’s, but will encounter the hairpin so, releases RNA from polymerase and polymerase from DNA • Rho dependent: this is a protein that recognizes nucleotides that act as termination signals for RNA polymerase o Do not have hairpin or string of U’s at 3’ end o 40-60 nucleotides rich in C’s, poor in G’s and have upstream rut site (rho utilization) o Bind nascent RNA chain at the rut site, upstream from sequences where RNA polymerase pauses o Rho factors facilitate release of RNA from polymerase o So: binding of rho to rut, pause, rho-mediated dissociation ^ Rho binding to rut 8.3 Transcription in Eukaryotes 1. Larger eukaryotic genome = more genes to be recognized and transcribed a. Much more non-coding DNA in eukaryotes; genes farther apart b. Makes initiation step much more complicated; for multicellular eukaryotes, finding the start of the gene can be very difficult i. Have 3 RNA polymerases for I – rRNA genes, II – protein encoding genes and III – small functional RNA’s ii. Require assembly of many proteins at a promotor before RNA poly. II can begin to synthesize RNA; some proteins called general transcription factors (GTFs) 2. Presence of a nucleus in eukaryotes a. Transcription and translation are spatially separated – transcription in the nucleus and translation in the cytoplasm b. Before it leaves the nucleus, primary transcript must be modified 3. DNA for transcription is organized into chromatin in eukaryotes (naked in prokaryotes) a. Regulates eukaryotic gene expression 15 BIOL205 NOTES Transcription Initiation in Eukaryotes - Starts when sigma subunit of RNA polymerase holoenzyme recognizes -10 and -35 o Subunit will dissociate afterward, but transcription continues inside bubble - Requires GTFs to bind to regions in promotor before binding of core enzyme o Do not take part in RNA synthesis o Include TFIIA, TFIIB etc. o Recognize and bind to sequences in promotor or to other GTFs and attract the RNA polymerase II core and position it at correct site to start transcription o GTFs and RNA polymerase constitute the preinitiation complex (PIC) - Chimeric RNA polymerase II complex o Sequence of AAs of some of the RNA polymerase II core subunits is conserved from yeast to humans – form chimeric complexes - Promotors located on 5’ side of start site - TATA box: ~30 base pairs from transcription start site o Binding of TATA binding protein begins transcription; part of TFIID; attracts GTFs and RNA polymerase II to form the PIC o Poly. degrades, some GTFs stay to attract next RNA poly. core - Beta subunit of RNA polymerase II has a carboxyl terminal domain (CTD) – protein tail – located near where new RNA will emerge from polymerase - Elongation begins after CTD has been phosphorylated by GTFs – weakens bond between poly. and other PIC proteins to allow elongation Elongation, Termination and pre-mRNA Processing in Eukaryotes - Inside transcription bubble - Must undergo further processing before it can be translated - Processing after RNA synthesis is complete is said to be post-transcriptional - Processing actually takes place during RNA synthesis so, cotranscriptional - CTD coordinates all processing events o Determines wh
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