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Biochemistry Test 3

Tri 2

James Watson and Francis Crick described nature od DNA and molecular biology was born
Genome genetic information in the sequence of DNA, one complete set of information(haploid), DNA is in the nucleaus and in mitochondria
Central Dogma DNA is either REPLICATED into more DNA or is TRANSCRIBED into RNA. RNA is then TRANSLATED into PROTEIN
Molecular Biology includes the molecular nature of genetic material and is how the information in DNA id transmitted(inheritance) or translated(gene expression).
Replication the process by which genetic material is duplicated to allow cell division within a generation and allow genetic information to flow from one generration to the next
Transcription the genetic message in DNA is transferred to RNA by the process of DNA directed RNA synthesis.
Reverse Transcription RNA directed DNA synthesis.
Transcription Products mRNA, tRNA, rRNA
Translation when the genetic message is decoded by translating a linear sequence of AA, also called RNA-directected protein synthesis
Structure of DNA double helix (two polynucleotides), antiparallel (oposite polarity ), nitrogenous bases oriented inwards
Structure of DNA 2 5' to 3' sugar phosphate backbone, B-form of RNA (hydrated form) has 1 Base Pairs /turn. Major and Minor groove. Entire structure stabalized by h-bonds between bases, base stacking innteractions, sugar phosphate backbone on outside interacting with H2O
DNA Replication introduced by Watson and Crick, model suggested a model for faithful duplication-semiconservative DNA replication. In bacteria the genophore is a single circle. In eukaryotes the chrms are linear and multiple
Nucleotides Monomers of Nucleis Acid synthesis. DNA bases are ATGC (deoxyribose). RNA bases are AUGC (ribose)
Polymerization produces a phosphodiester backbone. 5' phosphate in one end and a 3' OH on the other, Extensions add from 3' OH. The chain grow 5' to 3'
Watson-Crick base pairs A-T(2 H bonds produced) G-C . The aromatic nature of nitrogenous bases produces a flat planar interaction, and allows for van der Waals interactions referred to as a base stacking interaction
Denaturation of DNA The two strands of DNA molecule can be seperated by heating or by additions of acid or base. Unwinding the double helix is called melting and if induced by heating has a Tm. Seperated strands will reassociate or reanneal to form the double helix apon co
Antiparallel the sugar phosphate backbone has sugars running in opposite directions. 5' to 3' is reversed from one side to the other
Structure of DNA B- form of DNA is the most common, and is Hydrated. There are 1 base pairs per turn. The helical nature produces a majr and minor groove, these sites are where we can see the bases of DNA
A form DNA dehydrated
B form DNA hydrated
Z form DNA in stretches of DNA which are G-C rich repeats. Alexander rich proposed. Important in gene expression, forms an upstream tight rregion of DNA important in gene expression
DNA vs RNA in Alkali(stongly basic) In an alkaline solution, DNA will denature and RNA is broken down to SS and broken peices. A DNA-RNA hybrid has a RNA broken down leaving ssDNA, which is useful in the lab
DNA RNA interaction We use U instead of T in RNA
Eukaryotic DNA In the nucleaus, the structure beads on string, there are histone protein interactions,
Chromatin Structure H2A, H2b , H3 and H4 makeup bead. There are two copies of each, with a total of 8. There is also an H1 linker.
Homologous Chrms For examole, Chms #1 has a paternal and maternal part.
Homologs contain same basic data set of information, correspondence in a linear fashoin, potential for minor varients between the loci(alleles)
Structure of a gene Each gene has an upstream promoter region, this is important for letting the cellular apparatus know where to begin the transcription. RNA polymerases recognize this region. A portion of the RNA transcribed is the leader and not translated (mRNA)
Ribosome Prokaryotes =70s (50s =30s). Eukaryotes = 80s (60s + 40s) S= sedimentation rate
Retroviruses retro b/c they take the viral RNA and make a DNA copy. They use enzymes called Reverse Transcriptase and have the ability to integrate into the host chms. LTR (long terminal repeats) are used in the process
Central Dogma DNA is TRANSCRIBED into RNA and then the RNA is TRANSLATED into PROTEIN
DNA Polymerase for replication
RNA Polymerase transcription and translation
Replication of DNA achieved through DNA polymerase. Catalyzes the step by step additions of nucleotides to growing polynucleotide chain. Requires all four dNTP's (AGTC) and Mg+2. All additions are to the 3' OH group
Replication of DNA 2 The two strands must be seperated for replication, thus a replication fork is formed. Elongation of the newly synthesized polynucleotide is always 5' to 3'. The opposite strand serves as a template for which a new base will be incorporated
Replication of DNA 3 DNA ploymerase I was the first polymerase discovered. (ARTHUR KORNBURG). He found them in E. coli. The major function is to repair DNA damage, remove RNA primers. DNA polymerase III of E. coli is the major polymerase for the genophore
Replication is bi-directional JOHN CAIRNS did experiments showing the bi-directional nature of replication in E. coli. He labeled the genophore using 3H Thymine. Thymine only doesinto DNA, he termed what he saw as theta structures
Replication proteins note the following
Primosome complex (Prok and Euk) One component of the complex called PRIMASE synthesizes RNS primer
Helicase ( Prok & Euk) unwinds DNA dbl helix, costs 2 ATP/ nucleotide pair
Single Stranded Binding Protein (SSB) (Prok & Euk) stabalizes ssDNA @ replication fork
TopoisomerasesI Prok & Euk) allows strand to spin around, prevents tension
Topoisomerase II (Prok & Euk) DNA gyrase, imp in gene expression
DNA Polymerase I (Prok) Repairs and removes DNA
DNA Polymerase II (Prok) synthesis of bacteria
DNA Ligase (Prok & Euk) seals end of backbone (covalent link)
DNA Ploymerase alpha (Euk) proof readds, lagging strand synthesis
Telomerase (euk) extends length
Polymerase Reaction Each time a new base is added, DNA polymerase senses the proper fit by the size, and proper Watson-Crick pairing. There must always be enough dNTP and Magnesium
Steps in DNA replication The helicase first unwinds the parental strands, SSB's stabilize the ssDNA, Primosome Complex binds the ssDNA and synthesizes RNA complex. DNA poly III uses RNA Primer, but it requires a 3' OH group to befin the replication
Steps in the DNA Replication The RNA primers are removed from the DNA poly I, the new synthesized DNA fragments are joined by DNA ligase. Topoisomerase I relieves upstream tension and Topoisomerase II resolves concatamers.
Bruce Albert's Model Sliding Trobone model of DNA replication showing the arrangement for replication of the lagging strand.
Replication of Linear DNA telomeres of linear chrms are a problem, there is a mechanism to add to these ends so they do not shorten
Telomerase Function is to lengthen telomeresand prevent erosion of genetic information. When this slows, aging of the cell and cancers are thought to occur. Telomerase carries its own RNA primer to extend the ends of chrms (telemeres)
DNA Damage Any change in normal base sequence is a mutation. Some of these lead to cancerous cells. The carcinogen in cigarettes is Cenzol(alpha)pyrene witha GC base pair in DNA leading to Guanine
Excision Repair (Depurination Repair) repair endonucleases that induce a nick into the backbone. DNA pol I senses the nick and DNA ligase seals the backbone again.
Deamination fo cytosine to uracil no uracil is allowed in DNA, the enzyme Uracil-N-glycosylase ung removes uracil. Repair follows as in Depurination repair
Thymine Dimer Repair This is a big problem. Either a short patch (excision repair) or the enzyme called PHOTOLYASE can undo the cyclobutane ring b/tw intrastrand T dimers. PHOTOLYASE utilizes energy from visible light to do the rrepair, they are found in skin cells
Recombination This is at times when damage is great. SOS repair requires that this occur, this is also happening in meiosis when crossing over observed as chi forms are observed.
Recombination STEPS First One strand is nicked and strand invasion occurs. Then a D-loop nick is formed and is nicked which promotes base pair b/tw homologous regions. Ligation occurs and a HOLIDAY STRUCTURE is seen. Cut at 'a' then reseal or cut at 'b' and reseal.
Burkitt's lymphoma Also known as a Robertsonian translocation..Chrms # 8 ans chrms # 14.
Acute Polymphocytic Leukemia translocation at chrms # 15 and 17
Reverse Transcriptase making a DNA copy of a RNA molecule, this enzyme 1st seen in retroviruses.
Reverse Transcription Rxn There is 1st a copy of RNA template. Then a DNA-RNA hybrid is formed. The RNA is degraded by ribonuclease and leaves only DNA known as cDNA. The cDNA is synthesized into another strand forming dbls cDNA.
Transposons Found by Barbara McClinttock (Nobel Prize). Noted jumping genes which are direct repeats or long terminal repeats that are involved in a non-homologous pairing event. Multi colored corn
Transposons and Transposition rxn staggard breaks are produced by transposase, and insertion of transposon into recipient DNA. The direct repeats are important b/c they are at both ends. These insertions may interupt genes or change gene expression. May also remain quiet and show later
HIV hass these tranposons and transpositions
HIV a retrotransposon first requires reverse transcriptase to make RNA genome into DNA. Now the DNA is subject to tranposition. The DNA is then inserted into the chrms of infected cells, and each time it divides the viral DNA is replicated also
Transcription Synthesis of RNA
Regions of a Gene Promotor region( -#) closer to 5' end. Start point for transcription is +1, after promoter region. The Coding Region starts with a leader RNA and ends with a trailor RNA.
What is a gene? A segment of DNA that generateds an RNA product, or a protein product. The transcribed region contains the template for RNA synthesis, and begins at the start point (+1). A gene also includes an upstream promoter which regulates the production of the pr
Alpha connect subunits
Beta catalyze RNA Synthase
Beta ' Template binding and association with sigma
Sigma recognition of general promoters
Regulation of Transccription Basis if regulation is the prescence of conserved sequences upstream from the gene to be transcribed. Bacteria hae either a promoter region alone (Constitutive expression) or Promotor/Operator( inducible expression)
Prokaryote Model The initial contact point for RNA polymerase to bind is between -70 and -35. Between -7 and -10 is the TATaat box (Pribnow Box) or the promoter box. Transcription starts at +1.
Steps in Transcription Binding-recognitionof 5' upstream. Initiation-begin RNA synthesis(no primer). Elongation-continue polymer. Termintion-stop RNA synthesis.
Binding RNA holoenzyme binds DNA of promoter. sigma facto ris the important factor in recognition of the 5' end. Holoenzyme binds -35 first, then slides down to the pribnow box (-10) =, unwinds DNA and begins at +1
Initiation The beginning of RNA synthesis, 5' to 3' . First base is TRIPHOSPHATE. second is joined to 3' OH. Sigma dissosociates after 8-9 nucleotides
Elongation Polymerizarion continues, there is movement of core enzyme, requires enough ATP, CTP, GTP and UTP
Termination There are two types..Hairpin loop (GC loop) followed by AAAA in DNA and UUUU in RNA. Also Rho termination protein
Euk Transcription uses several RNA polymerases. more extensive upstream promoter. Modulation
Euk RNA Polymerases see next
Euk RNA Pol I in nucleolus, produces 35s and 47s preribosomal RNA
EUk RNA Pol II Nucleoplasm ..produces heterogenous nuclear RNA
Euk RNa Pol III Nucleoplasm..produces tRNA and more
Variation in Regulation more upstream DNA involved. Cis elements are needed close and Trans are far away.
RNA Pol III promoters for RNA Pol III are found within the coding exons.
Differences in gene expression Prokaryotes -Operons, constitutive and inducible, coupling transcription /translation. Euk- Monocistronic, Intons/Exons, 3 RNA pol, extensive RNA modification, slower gene expression, T/T seperated
Prokaryotes gene expression optimized to change quickly and adapt to env. changes. RNA produced has a half life(3 sec to a minute)
Eukaryotes gene expression complex, slower, long half life( DAYS TO MONTHS)
Amanita phalloides mushroom that produce a toxin alpha amanitin. Death cap. this toxin blocks RNA Pol III. 40-90% die within a few days.
mRNA Prok-no processing. Euk-primary transcript hnRNA.
hnRNA contians transcript for both exons and introns.
spliceosome cuts out introns and splices exons togetherin frame. made up of small nuclear RNA called U1, U2 etc. an dsmall snRNP's
Lariat Processing A common form of hnRNA processing. Conserved sequence observed at 5' end of exon/intron junction. These tell U RNA where to cut and where to splice
7 methyl G cap joined to 5' end in an unusual 5' to 5' linkage. Ribose sugars in the end may be methylated too. This structure is resistant to 5' exonucleases and this promotes stability of messsage
Poly A Tail Unusual Structure at 3' end. 40-200 A residues(Adenosine) are added post transcriptionally. Their function is to further stabalize the messaga against 3' exonucleases. It also facilitates exit from the nucleus.
mRNA Synthesis 7 me G Cap first, Poly A tail next, Splicing third, release from nucleus to cytosol
RNA processing Physical and Chemical
Physical RNA processing cleavage and loss of parts of RNA, cleavage and rejoining portions of RNA
Chemical RNA processing additions of nucleatides, modifications of existing nucleotides
rRNA genes found at nucleolus, segmental DNA contains tandem repeats in addition the DNA is amplified. 48% is spacer RNA in transcript, 52% is remainder of 18s rRNA
tRNA Removal of 5' leader seq., cut 3' OH nucleotides off and replace with CCA3'OH, extensive modifications to remaining selective bases, euk have also to splice out introns
RNA synthesis inhibitors by binding DNA or RNA Pol.
DNA Binders Actinomycin D
RNA Binders Rifampin, streptolydigin, Euk alpha amanitin
Highly Repetitive DNA 6-100 pairs, millions of copies, clustered locations, centromers, telomers, 10% of human genome not Transcribed
Moderate Repetitive DNA few to 10000 copies, 25% of human genome, transcribed by rRNA, tRNA histones
Translation protein synthesis, protein is final structure, requires all 3 forms of ribosome and magnesium, potassium
Basis of Translation A gene is usually over 1,000 nucleotides long, the nucleotides are trancribed to RNA. Codons are triplets of nucleotides
Genetic Code based on triplet or codon. The code has a reading frame, and is non-overapping. There are multile codons for the same AA
Point Mutation a single base is changed in the DNA producing a single change in RNA
Silent Mutation do not affect AA seq.
Missense Mutation one AA is replaced by another AA in the protein sequence
Nonsense Mutation premature termination of chain, change from codon to a stop codon
Wobble Hypothesis helps to account for degeneracy of the code. H- bonding at the first and second positions of the codon are normal Watson-Crick pairs. H bonds at the third is flexible- the base Inosine in anticodon can bind with U, A and C
GC Characteristics there is only 1 codon for 2 AA. AUG in mRNA codes for methionine. All proteins start with meth. UGG codes for tryptophan, no other codon. UAA, UAG and UGA are nonsense codons, they stop translation
Components of Translation Amino-acyl-tRNA synthetases, these are enzymes that charge the tRNA with their AA.Requires two high energ P from ATP. forms Amp
Energy expediture energy is used in formation of initiation complex, loading incoming charged tRNA and everthing else
Codon Recognotion via H-bonding to the anticodon of charged tRNA
Steps in Translation Initiation Complex, Elongation, Termination
Initiation Complex 30s ribosomal subunit finds Shine-Dalgarno sequence in the 5' end of mRNA. Initiato tRNA H-bonds with AUG in P site. 50s Ribosomal subunit joins complex
Initiation Factors must be present
Replication the process by which genetic material is duplicated to allow cell division within a generation and allow genetic information to flow from one generation to the next
Transcription The process when genetic message in DNA is transferred to RNA by the process of DNA DIRECTED RNA SYNTHESIS . The transcription products are mRNA, tRNA, rRNA
Translation the genetic message is decoded by translatinf a linear sequence of nucleotides into a linear seq. of AA
Watson and Crick DNA model and semiconservative replication
B form DNA most common, 10 base pairs per turn, major and minor groove(where you can see the nitrogenous basis)
Z form DNA Found by Alexander Rich, stretches of DNA that are GC rich, forms an upstream tight region
Sugar Phosphate backbone found on the outside
Gene expression upstream region
Alkali Solution and DNA DNA denatures
Alkali Solution and RNA RNA breaks down into peices of mononucleotides
Chromatin Structure contains H2A, H2B, H3 and H4. There are 2 of each, and an H1 linking them all
Homologous Chrms contain paternal and maternal parts
Prokaryote Ribosomes 70s (50s+30s)
Eukaryote Ribosomes 80s (60s +40s)
Retroviruses they take viral RNA and make a DNA copy by using enzyme reverse transcriptase. LTR's are used in this process
Arthur Kornberg discovered Polymerase I, found in E. coli cells. Poly III of E. coli is major poly for the genophore
John Cairns showed bidirectional nature of E. coli. Isolated a theta structure
Primosome Complex (prok and euk) synthesizes RNA primer
Helicase (prok and euk) unwinds DNA dbl helix, costs 2 ATP
SSB (prok and euk) stabalizes ssDNA at replication fork
Topoisomerase I (prok and euk) allows strand to spin around, prevents tension
Topoisomerase II (prok and euk) DNA gyrase, imp in gene expression, resolves concantamers`
DNA Poly I (prok only) Repairs and removes DNA
DNA Poly II (prok only) synthesizes bacteria
DNA Ligase (prok and euk) seals end of backbone (covalent link)
DNA Poly alpha (euk) proof reads lagging strand
DNA Poly sigma (euk) synthesizes leading strand
Telomerase (euk) extrends length at end
S phase Replication of DNA
Bruce Alberts sliding Trombone model, shows replication of lagging strand
Telomerase (blank)
Cigarette Carcinogen Benzol(alpha)pyrene
Excision Repair repair endonucleases and induce a nikc into backbone, DNA Poly I senses nick, DNA ligase seals backbone
Thymine Dimer Repair same as excision repair. Photolase is backup
Reverse Transcriptase Rxn Copy RNA template, RNA-cDNA hybrid formed, Degradation of RNA by ribonucleotides, cDNA synthesizedtoi make dbl stranded cDNA
Barbara McClintock discovered Transposons
Transposons jumping genes, direct repeats, this is the action in HIV
HIV retrotransposon, jumping genes
RNA poly's quaternary structure, 2 alpha units, two beta units and a sigma unit. The sigma unit makes it a holoenzyme
Upstream Region Promoters are AT rich, Pribnow Box (TATAAT) at -10. Transcription begins at +1.
Termination hairpin loop-(GC loop) folowwed by AAAA in DNA and UUUU in RNA . Also Rho termination protein
Euk RNA Polymerases (blank)
Pol I in nucleolus
Pol II in nucleoplasm
Pol III in nucleoplasm
CATT and TATA(hogness box) cis acting elements
RNA Pol III found within codong exons
Prok Gene Expression operons-polycistronic mRNA, Constitutive and inducible, coupling transcription/translation
Euk Gene Expression monocistronic, Introns/exons, 3 RNA Poly's, extensive RNA modification, slow changes in gene expression, T/T seperated
Amanita phalloides mushroom producing alpha-amanitin toxin, death cap, blocks RNA poly II, most will die
Lariat Processing a form of hrRNA, conserved sequence at 5' end of exon/intron junction, signals used by U RNA to recognize where to cut ......5' to 2' ester linkage formed
7methyl G cap 5' to 5' linkage, promotes stability of message, in Euk mRNA
Poly A Tail Euk mRNA at 3' end, additional Adenosine residues 40-200 added. Stabalizes seq., facilitates exit from nucleus
Mutations any change from normal coding sequence of the DNA, a nucleotide change
Point mutation a single base is changed in the DNA producing a single change in the RNA
Silent Mutation these do not affect the AA seq.
Missence Mutation 1 AA is replaced by another AA inthe protein seq.
Nonsense mutation premature termination of a polypeptide chain, change from codon to stop codon
Wobble Hyp to account for degeneracy of a code, H-bonding in 3rd position is flexible, the base Inosine in anticodon can bind with U, C and A
AUG in mRNA codes for methionine
Met all proteins start with this
UGG codes for tryptophan, no other codon for trp
UAA, UAG, UGA nonsense codons, stop translation
Euk General Cap at 5' end binds eIFs and 40s ribosomal subunit+tRNA met mRNA is scanned for AUG. 1st AA= methionine. Initiation factors=eIFs(12+) Ribosomes= 80s
Prok General Shine-Delgarno seq. upstream of AUG binds complementary 16SrRNA. 1st AA=Formyl-Met. Initiation Factor=Ifs(3) Ribosomes=70s
Antibiotics See old tests..matching
3 fates of RER Proteins Secretion, Lysosome, Membrane Glycoprotein
Streptomycin binds 30S ribosomal subunit of prok. Prevents initiation
Tetracycline binds 30s ribosomal subunit and inhibits binding of aminoacyl tRNA to A site
Chloramphenicol binds to 50s and inhibits peptidyltransferase
Erythromycin binds to 50s and prevents translocation
Created by: hwhite