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From Gene to Protein. Campbell and Reece Chapter 17. Gene Expression. process by which DNA directs the synthesis of proteins or RNA synthesis of proteins transcription translation. How Gene to Protein Figured Out. Evidence from study of metabolic disorders:

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From Gene to Protein

Campbell and Reece

Chapter 17


Gene Expression

  • process by which DNA directs the synthesis of proteins or RNA

  • synthesis of proteins

    • transcription

    • translation


How Gene to Protein Figured Out

  • Evidence from study of metabolic disorders:

  • 1902: British physician 1st to suggest genes responsible for phenotype thru enzymes that catalyze specific chemrx in the cell


Inborn Errors of Metabolism

  • Garrod hypothesized that symptoms of an inherited disease are due to a gene that leads to inability to make a certain enzyme

  • 1 of 1st to realize Mendel’s principle’s of heredity applied to more than pea plants


Alkaptonuria

  • Signs & Symptoms:

    • urine turns black when alkapton (chemical in urine) reacts with air

    • missing enzyme in pathway that degrades phenylalanine (a.a.)


Beadle & Tatum Experiment

  • worked with a bread mold Neurosporacrassa

  • bombarded it with radiation (already known to cause genetic changes)

  • then checked for survivors who had different nutritional needs from wild-type mold


Beadle & Tatum Experiment

  • individually put yeast in different mediums (agar with different nutrients)

  • identified mutants that could not survive on minimal nutrients placed them in complete growth medium (minimal med. + all 20 a.a. + few vitamins & minerals)


1-Gene-1-Polypeptide

  • Beadle & Tatum’s results supported their hypothesis

  • 1958: Nobel prize


1-Gene-1-Polypeptide

  • revised over time:

    • not all proteins are enzymes

    • some proteins have >1 polypeptide

    • now: 1- gene-1-protein hypothesis

    • not 100%: some eukaryotic genes can each code for a set of closely related polypeptides via alternative splicing


Transcription: short version

  • the synthesis of RNA using information in DNA

  • mRNA made using complimentary base pairing


Translation: short version

  • synthesis of a polypeptide using the information in mRNA

  • “translates” message in mRNA  a.a.


The Genetic Code

  • 4 nucleotide bases to code for 20 a.a.

  • triplet code: 3 consecutive bases code for 1 of the a.a./ stop


Template Strand

  • during transcription:

    • DNA helix unwound

    • 1 strand only transcribed (could be either side depending on the gene)


mRNA

  • uracil added as compliment to adenine

  • ribose as its 5-carbon sugar

  • single stranded


Codons

  • nucleotide triplets of DNA or mRNA that specifies a particular amino acid or termination signal

  • basic unit of the genetic code

  • written in 5’  3’ direction (in DNA 3 bases read in 3’  5’ direction)


Genetic Code


Cracking the Code

  • early 1960’s

  • Nirenberg: synthesized mRNA using only uracil (UUUUUUU…)

    • added it to test tube with all 20 a.a., ribosomes

    • translated into polypeptide made up of phenyalanine

    • now knew UUU = Phe

    • did same for AAA= Lys, CCC = Pro, GGG = Gly


Cracking the Code

  • all 64 a.a. deciphered by mid-1960’s

  • 3 codons code for “stop” marking end of translation

  • AUG functions as “start” & Met

    • Met may or may not be clipped off later


Genetic Code is Redundant

  • >1 triplet codes for each of the a.a. but any 1 triplet codes for only 1 a.a

  • redundant triplets usually only differ in the 3rd base


Reading Frame

  • translating the code in correct groupings

  • example:

    Did the red dog eat the bug?

    Idt her eddogeatthebug?


Reading Frame


Evolution of Genetic Code

  • code is nearly universal:

    • bacteria  complex multicellular organisms CAU = His

    • insert genes into other species & get same result (human insulin gene in bacteria)

  • exceptions: certain unicellular eukaryotes & in organelle genes of some species


RNA Polymerase

  • unwinds 2 strands of DNA

  • binds nucleotides together as build mRNA

    • only in 5’  3’ direction (like DNA polymerase)


3 Stages of Transcription

  • Initiation

  • Elongation

  • Termination


Initiation

  • After RNA polymerase binds to promoter, ¤ DNA strands unwind

    • polymerase begins RNA synthesis @ start pt. on template strand


Initiation

  • promoter: usually includes w/in it the transcription start point (a nucleotide where transcription begins) & extends several dozen or more nucleotide pairs upstream from start pt.

  • RNAP can assemble nucleotides only in 5’  3’ direction (just like DNA polymerase)

  • unlike DNAP, RNAP does not require a primer


Start Point

  • nucleotide where RNA synthesis actually begins

  • RNAP binds in precise location & orientation on the promoter  where determines where transcription starts & which of the 2 strands will be transcribed


RNA Polymerase

  • Bacteria:

    • 1 single RNAP used to make all types RNA

  • Eukaryotic Cells:

    • @ least 3 types RNA polymerase

    • II used for RNA synthesis

    • I and III used to transcribe RNA not used for protein synthesis


RNA Polymerase

  • Prokaryotes :

    • RNAP recognizes & binds to the promoter by itself

  • Eukayotes:

    • collection of proteins , transcription factors, mediate the binding of RNAP & initiation of transcription


Transcription Factors

  • must 1st attach to promoter b/4 RNAP II can bind to it

  • RNAP II + transcription factors =

    Transcription Initiation Complex

  • TATA box: DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex


Elongation

  • RNAP moves downstrean, unwinding the DNA & elongating the RNA transcript 5’  3’

  • ~ 10 – 20 nucleotides exposed

  • in wake of transcription the 2 DNA strands spontaneously rewind

  • length of DNA transcribed = transcription unit


Elongation


Termination

  • mechanism differs between prokaryotes & eukaryotes

  • Bacteria: transcription proceeds thru terminator sequence in the DNA  the transcribed RNA functions as the terminator sequence  causing RNAP to detach

  • prokaryotes have no further modification


Termination in Eukaryotes

  • RNAP II transcribes a portion of DNA called the polyadenylation signal (AAUAAA) in the pre-mRNA

  • ~10 – 35 nucleotides downstream from that sequence proteins ass’c with transcription cut the pre-mRNA free from the polymerase

  • pre-mRNA then  modified


RNA Processing

  • in eukaryotes only

  • both ends of primary transcript altered

  • certain interior sections cut out & remaining parts spliced back together


mRNA Ends

  • 5’ end receives a 5’cap: modified G is added after ~ 20 – 40 nucleotides in mRNA

  • 3’ end modified: enzyme adds 50 -250 A’s to the AAUAAA forming a poly-A tail


Functions of Modified Ends of mRNA

  • facilitate exit of mRNA from nucleus

  • protect mRNA from degradation of hydrolytic enzymes

  • help ribosomes attach to the 5’ end


RNA Splicing

  • cut-and-paste job removing segments of RNA that were transcribed

  • average size transcript: 27,000 nucleotides

  • average size protein: 1,200 nucleotides (400 a.a.)


Introns

  • noncoding, intervening sequence w/in primary transcript that is removed from the transcript during RNA processing; also refers to the region of DNA from which this sequence was transcribed


Exons

  • sequence w/in primary transcript that remains in the RNA after RNA processing; also refers to the region of DNA from which this sequence was transcribed


RNA Splicing

  • signal: short nucleotide sequence @ each end of an intron

  • particle called “snurp” recognizes splice sites

    • small nuclear ribonucleoproteins (snRNP’s)

    • in nucleus

    • made of RNA + protein

    • small nuclear RNA ~150 nucleotides


Spliceosome

  • combination of several different snRNP’s (almost size of ribosome)

  • interact with certain sites along intron releasing intron rapidly degraded

  • then joins ends of exons together


RNA Splicing


Ribozymes

  • RNA molecules that function like enzymes in some organisms

  • intron RNA can act like ribozyme & catalyze its own excision


Ribozymes

  • 3 properties of RNA enables some RNA molecules to function as enzymes:

  • single-stranded: 1 sequence can interact w/another using base pairing

  • some of bases contain functional groups (like a.a) that could participate in catalysis

  • ability to form H-bonds adds specificity


RNA


Importance of Introns

  • still having debate about importance of introns & RNA splicing in evolution

  • they both have adaptive benefits

  • do not know functions of most introns


Importance of Introns

  • single gene can encode >1 kind of polypeptide

  • know many genes that make 2 or more different polypeptides depending on what was removed as introns during gene splicing

  • called: alternative RNA splicing


Alternative RNA Splicing

  • Drosophila sex differences due to how RNA transcript is spliced

  • Human Genome Project: 1 of reasons humans get by with same # genes as a nematode


Translation: Closer Look

  • tRNA: transfers a.a. from cytoplasmic pool of a.a to ribosome where it’s a.a. is added to polypeptide chain

  • cell keeps supply of all 20 a.a. on hand

    • degradation of other molecules

    • synthesizes them using building blocks in cytoplasm


tRNA

  • brings specific a.a to ribosome

  • 1 end has a.a./ other end has anticodon which H-bonds with codon on ribosome

  • tRNA translates the codes into the corresponding a.a.

  • tRNA is transcribed from DNA templates & used repeatedly

  • tRNA made of ~80 nucleotides long with some regions folded back on self due to base pairing


tRNA Structure


tRNA Structure

  • 3’ end: a.a. attached

  • opposite end: anticodon


Accurate Translation

  • requires 2 instances of molecular recognition:

  • tRNA that binds to particular a.a.

    • correct match made by group enzymes called aminoacyl-tRNAsynthetases: their active site fits only 1 of the 20 a.a.

  • pairing of tRNAanticodon with mRNA codon


tRNA Wobble

  • ~ 45 different ones (not 61 like genetic code would suggest)

    • possible because pairing the 3rd base of codon & 3rd base of anticodon: relaxed base pair rules

      • U can pair with A or G in 3’ end of codon (3rd position)

      • called a “wobble”


Ribosomes

  • subunits made in nucleolus

    • rRNA transcribed & added to proteins imported from cytoplasm

    • ribosomal subunits  cytoplasm, join only when translating mRNA

    • subunits ~1/3 protein & 2/3 rRNA

      • bacteria: 3 molecules rRNA

      • eukaryotes: 4 molecules rRNA


Ribosome Structure

  • eukaryotic ribosomes slightly larger than prokaryotic ones

  • pharmaceutical products (antibiotics) designed to inactivate bacterial ribosomes that have no effect on ours

    • Tetracyclines

    • Streptomycin


Ribosome Structure

  • 4 binding sites: (1st for mRNA, others for tRNA)

  • mRNA binding site

  • P site: peptidyl-tRNA holds the tRNA carrying the growing polypeptide chain

  • A site: aminoacyl-tRNA holds tRNA carrying next a.a to be added

  • E site: exit, where discharged tRNAs leave ribosome


Ribosome

  • holds tRNA & mRNA in close proximity & catalyzes the formation of new peptide bond holding the 2 a.a together adding to carboxyl end of last a.a. in growing polypeptide chain

  • peptide chain passes thru exit tunnel in large subunit as it grows longer


Translation

  • 3 Stages:

  • Initiation

  • Elongation

  • Termination


Initiation

  • small ribosomal subunit attaches to mRNA

  • downstream from this attachment is the start codon AUG

  • tRNA with UAC (Met) binds to it

  • large ribosomal subunit attaches (1GTP)

  • initiation factors (proteins) required to bring it all together


Elongation

  • Codon recognition

    • anticodon of incoming tRNA w/c’ base

    • 1 GTP increases accuracy & efficiency

  • Peptide bond formation

    • part of rRNA catalyzes reaction

    • amino end of newest a.a + carboxyl end of peptide chain transferring pep. chain to tRNA @ A site

  • Translocation

    • ribosome moves so tRNA @ A site  P site

    • 1 GTP


Termination

  • ribosome reaches stop codon the A site accepts a “release factor” (shaped like tRNA but does not have aminoacyl part)

  • promotes release of bond between P site, mRNA, & last tRNA

  • 2 ribosomal subunits & ass’c proteins come apart


Animation Time!

  • http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html

  • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis


Try at home: interactive

  • http://www.wiley.com/college/boyer/0470003790/animations/translation/translation.htm


Polyribosomes

  • 1 ribosome can make polypeptide of average size: 1 min

  • typically many ribosomes are translating a single mRNA @ given time

  • 1st ribosome gets far enough past start codon 2nd ribosome can get started

  • allow cell to make many copies of polypeptide very quickly


Polyribosomes


Primary Structure

  • as polypeptide chain grows longer from ribosome it will spontaneously start to fold & coil as result of a.a side chain interactions

  • genes determine 1’ structure which then determines 2’, 3’ and 4’ structures


Chaperonins

  • proteins that help with the folding


Post-Translational Modifications

  • additional steps that may be required b/4 protein can do its job

    • attachment of sugars, lipids, phosphate groups to a.a

    • enzymatic removal of 1 or more a.a. from leading end (amino end)


Modification of Insulin


Targeting Polypeptides  Specific Locations in Cell

  • free ribosomes make proteins used in cytoplasm

  • bound ribosomes (RER) attached to cytosolic side while polypeptide being released into endomembrane system

  • both have identical small & large subunits


Ribosomes


Signal Peptide

  • growing polypeptide cues ribosome to attach to ER

  • polypeptides of proteins destined for endomembrane system have signal peptide: sequence of ~20 a.a. at or near leading end (N-terminus) is recognized by a protein-RNA complex called signal-recognition particle or SRP


SRP

  • escorts ribosome to receptor protein on ER membrane

  • receptor part of multiprotein translocation complex

  • ribosome continues to make polypeptide which enters ER thru protein pore

  • signal protein usually removed by enzyme


Proteins  Organelles

  • use other signal peptides for protein destined for chloroplast, mitochondria, or interior of nucleus

  • in these, proteins made in cytosol then  to organelle

  • signal proteins target or “address” proteins for secretion or to cellular locations

    • used by prokaryotes too


Mutations

  • ultimate source of new genes

    • large scale mutations

      • chromosomal rearrangements: chap. 15

    • small scale mutations

      • 1 or a few nucleotide bases changed


Try @ Home

  • http://www.bodrum-hotels.com/gene-mutations/gene-mutations-and-proteins-worksheet.html


Point Mutations

  • changes in single nucleotide pair

  • if occurs in gamete or cell that  gamete will be passed on to offspring

  • if mutation has adverse effect on phenotype is called a genetic disorder or hereditary disease

  • if mutation causes organism to die before fully developed it is said to be lethal

  • if mutation results in no change in phenotype is said to be silent


Sickle Cell Anemia


Familial Cardiomyopathy

  • point mutation

  • dominant

  • possible cause of sudden death of young athletes


Substitutions

  • replacement of 1 nucleotide pair by another pair: a few will improve activity of protein it is coding for but most will be detrimental

  • some silent due to redundancy of genetic code

  • if changes 1 a.a. for another called missense mutation

    • if substituted a.a. similar to real one no effect

    • some substitutions will have major consequences


Nucleotide-Pair Substitutions


Nucleotide-Pair Substitution

  • Silent

  • Missense:

    • most substitutions in this category

  • Nonsense: substitution changes from 1 a.a.  stop codon

    • resulting polypeptide is shorter

    • nearly all  nonfunctional proteins


Insertions & Deletions

  • (+) or (-) of nucleotide pairs in a gene

  • disastrous effects

  • may alter reading frame triplet codon shifts on mRNA

  • called frameshift mutation

    • whenever insertion or deletion not in a multiple of 3

    • if not causes major missense


Mutagens

  • any chemical or physical agent that interacts with DNA & can cause a mutation

  • 1920’s: Muller used x-rays to make mutant Drosophila & he discovered it does same in humans

  • mutagenic radiation includes:

    • UV radiation cause thymine dimers in DNA


Thymine Dimers


Chemical Mutagens

  • nucleotide analogs

    • similar to normal DNA nucleotides

    • insert self into DNA


Chemical Mutagens

  • some cause chemical changes in bases that changes their pairing properties


How Mutagens Determined


Gene Expression in 3 Domains


Differences

  • some in gene expression among eubacteria, archaea, and eukaryotes

  • if no nucleus: translation can begin b/4 transcription is over

  • Archaea show similarities to Eubacteria and eukaryotes in processes of gene expression


What is a Gene?

  • region of DNA whose final functional product is either a polypeptide or an ENA molecule


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