Biomol Part 2 Dr. Dani

8/10/2019 Biomol Part 2 Dr. Dani

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Airin Aldiani PD-A 2009 (0910710030) 1

Transcription maschineTranscription maschine

MOLECULAR BIOLOGYMOLECULAR BIOLOGY

Part IIPart II


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ContentContent

TranslationTranslation

MutationMutation

DNA repair systemDNA repair system

Manipulation on DNA and proteinManipulation on DNA and protein

TranslationTranslation

the whole process by whichthe whole process by which the nucleotidethe nucleotidesequence of an mRNA is used to order and tosequence of an mRNA is used to order and to

join the amino acids in polypeptide chainjoin the amino acids in polypeptide chain..

TRANSLATIONTRANSLATION

Component ofComponent ofTranslation:Translation:--mRNAmRNA--tRNAtRNA--RibosomRibosom

COMPONENTS OFCOMPONENTS OFTRANSLATIONTRANSLATION

1.1. mRNAmRNA

carries the genetic information transcribedcarries the genetic information transcribedfromfrom DNA in the form of a series of codonsDNA in the form of a series of codons..

2. tRNA.2. tRNA.

Each AA has its own subset of tRNAs, whichEach AA has its own subset of tRNAs, whichbind the AA and carry i t to the growing end ofbind the AA and carry i t to the growing end of

a polypeptide chain if the next codon in thea polypeptide chain if the next codon in themRNA calls for it. Each specific tRNA moleculemRNA calls for it. Each specific tRNA moleculecontainscontains anticodonanticodon, that can base, that can base--pair withpair withitsits complementary codoncomplementary codon in the mRNAin the mRNA


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tRNAtRNA

- a hairpin structure

- amino site at (3 end)

- anticodon site, that bind withcodon in mRNA

There are 61 type tRNA in cell

3.3. rRNArRNA

-- associates with a set of protein to formassociates with a set of protein to formribosomesribosomes -- move along an mRNA molecule,move along an mRNA molecule,catalyze the assembly of AA intocatalyze the assembly of AA into polypeptidepolypeptidechainschains..

-- Ribosomes are composed of a large andRibosomes are composed of a large andsmall subunit, each of which contains its ownsmall subunit, each of which contains its ownrRNA molecules.rRNA molecules.


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Translation ProsesTranslation Proses

InitiationInitiation

ElongationElongation

TerminationTermination

INITIATIONINITIATION

Binding of mRNA onBinding of mRNA onsmall subunitsmall subunitribosomribosom

Binding of tRNABinding of tRNAinisiator at the P siteinisiator at the P siteof ribosomof ribosom

Binding of bigBinding of bigsubunit ribosomsubunit ribosom


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ELONGATIONELONGATION

- Codonrecognition

- Peptide bondformation

- translocation

TERMINATIONTERMINATION

THE LAST STEP OF TRANSLATION

RIBOSOME (SITE A) REACH STOP CODON

(UAA UAG UGA)


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In eukaryotic cells, protein synthesis occurs in theIn eukaryotic cells, protein synthesis occurs in the

cytoplasm, where three types of RNA molecules comecytoplasm, where three types of RNA molecules cometogether to perform different but cooperative functions:together to perform different but cooperative functions:

MUTATIONMUTATION

MUTATION,MUTATION,

Type of mutation:Type of mutation:

1.Base pair (nucleotide pair) substitutions1.Base pair (nucleotide pair) substitutions

2.Frameshift mutations2.Frameshift mutations

3.spontaneous mutation3.spontaneous mutation

1.Base pair (nucleotide pair)1.Base pair (nucleotide pair)substitutionssubstitutions

--transitionstransitions

(purine to purine or pyrimidine to(purine to purine or pyrimidine topyrimidine)pyrimidine)

--transversionstransversions

(purine to pyrimidine or pyrimidine to(purine to pyrimidine or pyrimidine topurine).purine).


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The consequences of baseThe consequences of basesubstitution mutationssubstitution mutations

1.1. silent Msilent M,,not resulting in a new aa (amino acid) innot resulting in a new aa (amino acid) inthe protein sequencethe protein sequence, eg. GC, eg. GCAA or GCor GCGG..

2.2. missensemissense mutationmutation..result in an aa substitutionresult in an aa substitution ex. Cex. CTTCCCCAACCin the DNA sense strand (glutamatein the DNA sense strand (glutamatevaline).valine).

Ex.in the betaEx.in the beta--globin protein chain causingglobin protein chain causing sicklesickle--cellcellanemiaanemia,, hemoglobin Chemoglobin C oror no phenotypeno phenotype..

3.3. aa nonsensenonsense mutationmutationmutate an amino acid codon to amutate an amino acid codon to atermination codon or vice versatermination codon or vice versa..

4. Mutation occurs4. Mutation occurs in promoters or 5'in promoters or 5'regulatory regions of genes or in intronsregulatory regions of genes or in intronsmay affect their transcription, translation,may affect their transcription, translation,or splicingor splicing.. Ex.Ex.betabeta--thalassemias,thalassemias,result of nonresult of non--structural mutations affect the level of expression of thestructural mutations affect the level of expression of theglobin genesglobin genes..

Spontaneous frameshiftSpontaneous frameshift mutations 5'mutations 5'

ex.ex. AGAGTCAATCCATGAAAAAATCAG 3'TCAATCCATGAAAAAATCAG 3'3'TCAGTTAGGTACTTTTTTAGTC 5'3'TCAGTTAGGTACTTTTTTAGTC 5'

frameshifts are the result of "frameshifts are the result of "slippedslipped

mispairingmispairing" between the template" between the templateDNA strand and the newlyDNA strand and the newlysynthesized strand during DNAsynthesized strand during DNAreplicationreplication

2.Frameshift mutations2.Frameshift mutations3.Spontaneous mutation3.Spontaneous mutation

-- DNA replication errors and polymeraseDNA replication errors and polymeraseaccuracyaccuracy

Mistakes in DNA replication where an incorrectMistakes in DNA replication where an incorrectnucleotide is added will lead to a mutation in thenucleotide is added will lead to a mutation in thenext round of DNA replication of the strand withnext round of DNA replication of the strand withthe incorrect nucleotidethe incorrect nucleotide

-- Base alterations and base damageBase alterations and base damageThe bases of DNA are subject to spontaneousThe bases of DNA are subject to spontaneousstructural alterations calledstructural alterations called tautomerizationtautomerization::


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contcont

spontaneous base degradationspontaneous base degradation..

TheThe deaminationdeamination of cytosine to uracil.of cytosine to uracil.

spontaneous DNA damagespontaneous DNA damage viavia alkylationalkylation

the addition of alkyl (methyl, ethyl, occasionallythe addition of alkyl (methyl, ethyl, occasionallyprophyl) groups to the bases or backbone ofprophyl) groups to the bases or backbone ofDNA.DNA.

AlkylationAlkylation can occur through reaction ofcan occur through reaction ofcompounds such as Scompounds such as S--adenosyl methionine withadenosyl methionine withDNA. Alkylated bases may be subject toDNA. Alkylated bases may be subject tospontaneous breakdownspontaneous breakdown oror mispairingmispairing..

REPAIR" mechanisms :REPAIR" mechanisms :

Direct repairDirect repair

Simple removal of damage.Simple removal of damage.

Excision repairExcision repair

Recognition by enzyme, followed byRecognition by enzyme, followed byexcision. Glycosylases, uracilexcision. Glycosylases, uracilglycosylaseglycosylase----enzyme which removesenzyme which removesuracil from DNA, AP endonuclease.uracil from DNA, AP endonuclease.


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mutation, repair and tumormutation, repair and tumor

MutationMutation--Changes in DNA sequenceChanges in DNA sequencedecrease productiondecrease production

ofof tumor supressor proteintumor supressor proteinincrease unregulated production or activation ofincrease unregulated production or activation of

oncoproteinsoncoproteins cancercancer..

Most cancerMost cancer lack one or more DNAlack one or more DNA--repair systemsrepair systems,,

Some repair mechanism introduce errors in the nucleotideSome repair mechanism introduce errors in the nucleotidesequencesequenceoncogenesisoncogenesis..

Inability of tumor cells to maintain genomic integrity leadsInability of tumor cells to maintain genomic integrity leadsto formation of ato formation of a heterogenous population of malignantheterogenous population of malignantcellscells..

Chemotherapy toward a single gene or group of geneChemotherapy toward a single gene or group of gene --ineffectiveineffective


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Inhibitor of protein or RNAInhibitor of protein or RNAsynthesissynthesis

MANIPULATING PROTEIN, DNA, RNAMANIPULATING PROTEIN, DNA, RNA

RESTRICTION ENDONUCLEASERESTRICTION ENDONUCLEASE

Type II restriction endonucleaseType II restriction endonuclease

Palindromic sequencesPalindromic sequences

Recognition site/cleavageRecognition site/cleavagesite/restriction sitesite/restriction site

Type of cut endType of cut end 55--phosphat extention (phosphat extention (EcoEcoRI)RI)

3hydroxyl extention (3hydroxyl extention (PstPst I)I)

BluntBlunt--end (end (PvuPvu II)II)

plasmidplasmid

Def:Def:

Cyclic, selfreplicatingCyclic, selfreplicatingDNA, doubleDNA, doublestranded,maintainedstranded,maintainedin the bacterial cell asin the bacterial cell asa extrachromosomala extrachromosomal

DNA.DNA.

Def:Def:

Cyclic, selfreplicatingCyclic, selfreplicatingDNA, doubleDNA, doublestranded,maintainedstranded,maintainedin the bacterial cell asin the bacterial cell asa extrachromosomala extrachromosomal

DNA.DNA.


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BasicBasicpriciplespriciplesof DNAof DNAcloningcloning

ininplasmidplasmidvectorvector

Human genomicHuman genomicDNA lybraryDNA lybrary

POLYMERASE CHAIN REACTIONPOLYMERASE CHAIN REACTION


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Biomol Part 2 Dr. Dani

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