the “central dogma” overview of dna dnamrnaproteins replication transcriptiontranslation...
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The “Central Dogma”The “Central Dogma”Overview Of DNAOverview Of DNA
DNADNA mRNAmRNA proteinsproteins
ReplicatioReplicationn
TranscriptiTranscriptionon
TranslationTranslation
EnzymesEnzymes
StructureStructure
MovementMovement
HormonesHormones
Gas exchangeGas exchange
Amino Acid Amino Acid StorageStorage
Protein SynthesisProtein Synthesis
How does DNA control the structure How does DNA control the structure and function of the cell?and function of the cell?
it makes proteins!it makes proteins!– Structure: collagen, elastin, keratinStructure: collagen, elastin, keratin– Enzymes: catalase, amylase, sucrase, etcEnzymes: catalase, amylase, sucrase, etc– Hormones: insulin, glucagon, etcHormones: insulin, glucagon, etc– Amino acid storage: albumin, ovalbumin, Amino acid storage: albumin, ovalbumin,
etcetc
What is similar about protein synthesis in What is similar about protein synthesis in prokaryotes and eukaryotes? What is prokaryotes and eukaryotes? What is different?different?
Protein Synthesis!Protein Synthesis!
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scription.html TranslationTranslation http://www.johnkyrk.com/DNAtran
slation.html
Notas – From Notas – From Gene to ProteinGene to Protein
Metabolism teaches us Metabolism teaches us about genesabout genes
Metabolic defects caused Metabolic defects caused by non-functional enzymeby non-functional enzyme
Studying metabolic Studying metabolic diseases suggested that diseases suggested that genes specified proteinsgenes specified proteins– PKYPKY– Alkaptonuria (black urine)Alkaptonuria (black urine)
Genes dictate the Genes dictate the phenotypephenotype
1 gene – 1 enzyme hypothesis1 gene – 1 enzyme hypothesis Beadle and Tatum – 1941Beadle and Tatum – 1941
– Compared different nutritional mutants Compared different nutritional mutants of bread mold, of bread mold, NeurosporaNeurospora
– Created mutations by X-ray treatments Created mutations by X-ray treatments X-rays break DNA)X-rays break DNA)
– Wild type grows on “minimal” media Wild type grows on “minimal” media (sugar)(sugar)
– Mutants require different amino acids Mutants require different amino acids because each mutant lacks a certain because each mutant lacks a certain enzyme needed to produce a certain enzyme needed to produce a certain amino acidamino acid
– Conclusion: Conclusion: Broken gene = non-Broken gene = non-functional enzymefunctional enzyme
1 gene – 1 1 gene – 1 enzyme enzyme hypothesishypothesis
Beadle and Beadle and Tatum – 1941Tatum – 1941
Problems with:Problems with:– One gene – one enzymeOne gene – one enzyme
not all proteins are enzymes, and not all proteins are enzymes, and they’re coded by genes toothey’re coded by genes too
– One gene – one proteinOne gene – one protein many proteins consist of several many proteins consist of several polypeptide, and each polypeptide polypeptide, and each polypeptide has it’s own genehas it’s own gene
One gene – one polypeptide?One gene – one polypeptide?
Defining a gene…Defining a gene…
““Defining a gene is problematic because Defining a gene is problematic because small genes can be difficult to detect, one small genes can be difficult to detect, one gene can code for several protein products, gene can code for several protein products, some genes code only for RNA, two genes some genes code only for RNA, two genes can overlap, and there are many other can overlap, and there are many other complications.”complications.” – Elizabeth Pennisi, – Elizabeth Pennisi, ScienceScience 20032003
How would YOU define a gene in your own How would YOU define a gene in your own words?words?
From nucleus to cytoplasm…From nucleus to cytoplasm…
Where are the genes? Where are the genes?
in DNA on chromosomes in the nucleusin DNA on chromosomes in the nucleus
Where are proteins synthesized?Where are proteins synthesized?
on ribosomes (free or on the ER) in the on ribosomes (free or on the ER) in the cytoplasmcytoplasm
How does the information get from the How does the information get from the nucleus to the cytoplasm?nucleus to the cytoplasm?
mRNA is made in the nucleus and can travel mRNA is made in the nucleus and can travel into the cytoplasm to the ribosomesinto the cytoplasm to the ribosomes
deoxyribose ribose
A-T, C-G T-A, A-U, C-G
Double Single
Transcription!Transcription!
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Transcription Basics1. Initiation
RNA polymerase binds to promoter sequence on DNA
1 where to start reading = Promoter (initiation site)2 which strand to read = template strand3 direction on DNA = reads 3’5 builds 5’ 3’
2 Elongation RNA polymerase unwinds DNA ~20 bp at a time Reads DNA 3’ 5’ Builds RNA 5’ 3’ No proofreading, about 1 error/105 bases Many copies, short life, no problem
3 Termination RNA polymerase stops at termination sequence mRNA leaves nucleus through pores
TranscriptiTranscriptionon
promoter
InitiationRNA
polymerase
Terminator
Elongation
Template Strand
mRNATerminatio
n
Completed mRNA transcript
RNA Processing or RNA Processing or EditingEditing 5’ cap5’ cap
– protectionprotection– targets mRNA for ribosometargets mRNA for ribosome
Poly-A tailPoly-A tail– protectionprotection– leads mRNA out of nucleusleads mRNA out of nucleus
SpliceosomeSpliceosome– composed of snRNPs (small nuclear composed of snRNPs (small nuclear
ribonucleoproteins)ribonucleoproteins)– introns – introns – intervening, interrupting = removed by intervening, interrupting = removed by
spliceosomespliceosome– exons – exons – expressedexpressed
SliceosomeSliceosome
snRNPs = small snRNPs = small nuclear nuclear ribonucleoproteiribonucleoproteinsns
snRNPs Other proteins
Spliceosome
Intron
Exons
AnimationsAnimations
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Putting it Together – Putting it Together – Transcription to TranslationTranscription to Translation
How does mRNA code for proteins?How does mRNA code for proteins? How can you code for 20 aa with only How can you code for 20 aa with only
4 nucleotide bases (A, U, G, C)?4 nucleotide bases (A, U, G, C)? How can an alphabet of 4 letters How can an alphabet of 4 letters
(nucleotides) translate into an (nucleotides) translate into an alphabet of 20 letters (aa)?!alphabet of 20 letters (aa)?!
Breaking the codeBreaking the code Nirenberg and MatthaeiNirenberg and Matthaei Determined 1st codon – amino acid matchDetermined 1st codon – amino acid match
– UUU coded for phenylalanineUUU coded for phenylalanine Created artificial poly(U) mRNACreated artificial poly(U) mRNA Added mRNA to test tube of ribosomes and Added mRNA to test tube of ribosomes and
nucleotidesnucleotides– mRNA synthesized a single amino acid polypeptide mRNA synthesized a single amino acid polypeptide
chain: phe-phe-phe-phe-phe-phechain: phe-phe-phe-phe-phe-phe
DNA
Gene 1
DNA3’ 5’
5’ 3’
Transcription
mRNA
Translation codon
Protein
Amino acids
The CODE!!The CODE!! For ALL life!! (yes, even prokaryotes…)For ALL life!! (yes, even prokaryotes…)
– Strongest support for common origin for all lifeStrongest support for common origin for all life Code is redundantCode is redundant
– Several codons for each amino acidSeveral codons for each amino acid Start codon: AUG = methionineStart codon: AUG = methionine Stop codons: UGA, UAA, UAG Stop codons: UGA, UAA, UAG
TranslationTranslation Ribosome reads mRNA in codonsRibosome reads mRNA in codons tRNA brings in correct amino acidtRNA brings in correct amino acid tRNA matches codon of mRNA = tRNA matches codon of mRNA =
anticodonanticodon Amino acids assembled into polypeptide Amino acids assembled into polypeptide
chain chain
tRNA StructuretRNA Structure ““clover leaf” structureclover leaf” structure
– anticodon on “clover leaf” endanticodon on “clover leaf” end– amino acid on 3’ endamino acid on 3’ end– anticodon written 3’ anticodon written 3’ 5’ to match 5’ to match
codons which are 5’ codons which are 5’ 3’ 3’
Aminoacyl tRNA Aminoacyl tRNA synthetasesynthetase
enzyme which bonds amino acid to enzyme which bonds amino acid to tRNAtRNA– endergonic reaction (does it require endergonic reaction (does it require
energy?)energy?)– ATP ATP AMP (how many phosphates do we AMP (how many phosphates do we
use?) use?) – Energy stored in tRNA-aa bondEnergy stored in tRNA-aa bond
UnstableUnstable
RibosomesRibosomes Facilitate coupling of tRNA anticodon to Facilitate coupling of tRNA anticodon to
mRNA codonmRNA codon– Organelle or enzyme?Organelle or enzyme?
StructureStructure– Ribosomal RNA and proteinsRibosomal RNA and proteins– 2 subunits: large and small2 subunits: large and small– A site (aminoacyl-tRNA site)A site (aminoacyl-tRNA site)
Holds tRNA carrying next amino acid to be Holds tRNA carrying next amino acid to be added to chainadded to chain
– P site (peptidyl-tRNA site)P site (peptidyl-tRNA site) Holds tRNA carrying growing polypeptide chainHolds tRNA carrying growing polypeptide chain
– E site (exit site)E site (exit site) Discharged tRNA leaves ribosome from exit siteDischarged tRNA leaves ribosome from exit site
Building a Polypeptide1. Initiation
Brings together mRNA, ribosome subunits, proteins and initiator tRNA
2 Elongation3 Termination
Release polypeptide “release protein” bonds to
A site Bonds water molecule to
polypeptide chain
PolyribosomesPolyribosomes Many ribosomes read single mRNA Many ribosomes read single mRNA
simultaneously making many copies of a simultaneously making many copies of a protein!protein!
Protein TargetingProtein TargetingSignal polypeptideSignal polypeptide
– ~20 aa at the beginning of the polypeptide~20 aa at the beginning of the polypeptide– Recognized by SRPs (signal recognition particles)Recognized by SRPs (signal recognition particles)– SRP brings polypeptide and ribosome to ER so that SRP brings polypeptide and ribosome to ER so that
polypeptide is secreted into the ER as it’s built.polypeptide is secreted into the ER as it’s built.
Destinations – other signal polypeptides used Destinations – other signal polypeptides used to targetto target– SecretionSecretion– NucleusNucleus– MitochondriaMitochondria– ChloroplastsChloroplasts– Cell membraneCell membrane– Cytoplasm Cytoplasm
Protein TargetingProtein Targeting
Comparing!Comparing!
MutationsMutations
Are all mutations bad?Are all mutations bad?
Do all mutations lead to changes Do all mutations lead to changes in amino acids?in amino acids?
Sickle Cell Anemia - Sickle Cell Anemia - MutationMutation
K-ras Oncogene – Point K-ras Oncogene – Point MutationMutation
MutationsMutations Point MutationsPoint Mutations
– 1 base pair change1 base pair change– Base-pair substitutionBase-pair substitution
Silent mutation: no amino acid change Silent mutation: no amino acid change because of redundancy in codebecause of redundancy in code
Missense: change amino acidMissense: change amino acid
Nonsense: change to stopNonsense: change to stop
MutationsMutations Insertions – adding base(s)Insertions – adding base(s) Deletions – losing base(s)Deletions – losing base(s) BOTH cause frameshiftBOTH cause frameshift
QuestionQuestion
Which mutations, point mutations Which mutations, point mutations or frameshift mutations, do you or frameshift mutations, do you think are more harmful and why?think are more harmful and why?
This weekend…This weekend…
Work on Quest! You should be Work on Quest! You should be able to answer everything except able to answer everything except the questions about gene the questions about gene regulation which we will do on regulation which we will do on Monday in classMonday in class
Quest will be due Monday at Quest will be due Monday at midnightmidnight
Gene RegulationGene Regulation In a nutshell…In a nutshell…
Prokaryotes regulate transcription through operons.Prokaryotes regulate transcription through operons.Area where RNA Polymerase binds
Area where repressor can bind to stop binding of RNA polymerase Segment of DNA containing
several genes, a promoter, and an operatorCodes for
repressor; gene is upstream or downstream from operator
Regulatory Gene
2 Kinds of Feedback2 Kinds of Feedback
Prokaryotes regulate transcription through operons.Prokaryotes regulate transcription through operons.
Area where RNA Polymerase binds
Area where repressor can bind to stop binding of RNA polymerase
Segment of DNA containing several genes, promoter, and operator
Too much product, stop; not enough, keep going!! Keep going if you can!
Regulatory gene = codes for repressor; gene is upstream/downstream from operator
2 Examples of Negative 2 Examples of Negative FeedbackFeedback
Prokaryotes regulate transcription through operons.Prokaryotes regulate transcription through operons.Area where repressor can bind to stop binding of RNA polymerase
Segment of DNA containing several genes, promoter, and operator
Too much product, stop; not enough, keep going!!
Keep going if you can!
•Default “on” because repressor not bound
•Product binds to repressor to “activate” and turn “off” transcription when enough product has been made
•Usually anabolic pathways (ex: trp)
Regulatory gene = codes for repressor; gene is upstream/downstream from operator
Prokaryotes regulate transcription through operons.Prokaryotes regulate transcription through operons.Area where repressor can bind to stop binding of RNA polymerase
Segment of DNA containing several genes, promoter, and operator
Too much product, stop; not enough, keep going!!
Keep going if you can!
•Default “on” because repressor not bound
•Product binds to repressor to “activate” and turn “off” transcription
•Usually anabolic pathways (ex: trp)
•Default “off” because repressor bound
•Inducer binds to repressor to “inactivate” and release the repressor and turn “on” transcription only when there is substrate to be broken down
•Usually catabolic pathways (ex: lac)
Regulatory gene = codes for repressor; gene is upstream/downstream from operator
Prokaryotes regulate transcription through operons.Prokaryotes regulate transcription through operons.Area where repressor can bind to stop binding of RNA polymerase
Segment of DNA containing several genes, promoter, and operator
Too much product, stop; not enough, keep going!!
Keep going if you can!
•Default “on” because repressor not bound
•Product binds to repressor to “activate” and turn “off” transcription
•Usually anabolic pathways (ex: trp)
•Default “off” because repressor bound
•Product binds to repressor to “inactivate” and release the repressor and turn “on” transcription
•Usually catabolic pathways (ex: lac)
•Presence of activator turns “on”
•Ex: lac with lactose and no glucose
Regulatory gene = codes for repressor; gene is upstream/downstream from operator
Summary of Prokaryotic Summary of Prokaryotic Gene Regulation: OperonsGene Regulation: Operons
• Negative Feedback– Repressible– Inducible
• Positive Feedback
Gene RegulationGene Regulation In a nutshell…In a nutshell…
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•Histone acetylation
•Acetylated = less bound = easier access
•DNA methylation
•methylated = more bound = less access
Before transcription; which genes are “on” and “off”
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•Histone acetylation
•Acetylated = less bound = easier access
•DNA methylation
•methylated = more bound = less access
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp away
•Aids in RNA polymerase binding
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•DNA methylation
•methylated = more bound = less access
•Aids in RNA polymerase binding
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp awayRNA
ProcessingmRNA
Degradation
Translational Regulation
Protein Processing/Regulati
on
After mRNA has been made
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•DNA methylation
•methylated = more bound = less access
•Aids in RNA polymerase binding
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp away
•Alternate splicing = different combos of exons are expressed (some can be removed)
•Essential in making antibodies
RNA Processing
mRNA Degradation
Translational Regulation
Protein Processing/Regulati
on
After mRNA has been made
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•DNA methylation
•methylated = more bound = less access
•Aids in RNA polymerase binding
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp away
•Poly-A tail/5’ cap
•3’ and 5’ UTR (untranslated region) = nucleotides before the start codon (AUG) and/or after the stop codon
•RNAi = RNA interference small interfering RNAs (siRNAs) and microRNAs (miRNAs) = bind to mRNAs and prevent them from being translated or trigger their degradation
RNA Processing
mRNA Degradation
Translational Regulation
Protein Processing/Regulati
on
After mRNA has been made
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•DNA methylation
•methylated = more bound = less access
•Aids in RNA polymerase binding
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp awayRNA
ProcessingmRNA
Degradation
Translational Regulation
Protein Processing/Regulati
on
After mRNA has been made
•Poly-A tail/5’ cap = affect assembly/binding of ribosome
•3’ and 5’ UTR (untranslated region) = nucleotides before the start codon (AUG) and/or after the stop codon
Eukaryotes regulate gene expression pre-transcription, Eukaryotes regulate gene expression pre-transcription, during transcription, and post-transcription.during transcription, and post-transcription.
•Controls access of RNA polymerase to promoter
•DNA methylation
•methylated = more bound = less access
•Aids in RNA polymerase binding
•Transcription Factors = proteins that help RNA poly binding at promoter
•Activation sites and enhancer proteins = also aid in RNA poly binding; 1000s of bp awayRNA
ProcessingmRNA
Degradation
Translational Regulation
Protein Processing/Regulati
on
After mRNA has been made
•Processing= some proteins need to get folded, spliced (parts cut off), or have groups added
•Degradation= all proteins need to be “marked” for degradation/to get broken down; most “marked” by ubiquitin and broken down by proteosomes
Summary of Eukaryotic Summary of Eukaryotic Gene RegulationGene Regulation
• Pre-transcriptional
• During transcription
• Post-transcriptional