the “central dogma” overview of dna dnamrnaproteins replication transcriptiontranslation...

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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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