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Ch. 17 From Gene to Protein

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Essential Question:

How does the genetic information carried in the DNA of genes synthesize proteins that give us our traits?

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Proteins are the link between genotype and phenotype

gene expression = the process by which DNA directs protein synthesis

two stages:transcriptiontranslation

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1909 ­ Archibald Garrod­genes give us the phenotypes via enzymes that catalyze chemical reactions

­ inherited diseases = inability of a person to make an enzyme

Ex. alkaptonuria­black urine due to alkapton, blackens on

exposure to air­ most people have enzyme to break it down­people with alkaptonuria have inherited the

inability to make that enzyme

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Beadle and Tatum ­ mid 1930's­worked with Neurospora crassa (bread mold)

­wild type survived on minimal medium

­mutants needed complete growth medium (minimal + nutrients and 20 amino acids)

­figured out metabolic defect by putting each mutant strain in minimal medium + one nutrient or amino acid

*came to conclusion that one gene­one enzyme hypothesis

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Beadle and Tatum experiment

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problem ­ not all proteins are enzymesex. keratin, insulin

came up with new idea:one gene­ one polypeptide hypothesis

however, some genes code for RNA that never translate into proteins

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Two major steps to go from DNA to Proteins:

I. Transcription­synthesis of mRNA from DNA template

­DNA provides a template for making RNA sequence

II. Translation ­ synthesis of polypeptide under directions from mRNA

­ translates mRNA sequence to amino acid sequence of polypeptide

­ happens at ribosomes

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Why is RNA used instead of just DNA?

­ provides protection for DNA and genetic information (original copy is left untouched)

­more copies of protein can be made at the same time

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Difference in prokaryote and eukaryote protein synthesis

­prokayotes ­ no nucleus so ribosomes are near DNA, can happen faster

­eukaryotes ­ have nucleus so info from DNA is modified first, then has to get out of nucleus to ribosomes outside nucleus; happens slower

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Difference between prokaryote and eukaryote protein synthesis

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How do the nucleotides from DNA get read?

triplet codes = three consecutive bases­64 possible code words ­ specify 20 amino

acidsEx. AGU codes for serine

­in DNA only one strand codes for proteins = template strand (each strand may act as a template for certain genes)

­for a given gene the same strand is used all of the time

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RNA strand is complementary to DNA using the base pairing rules

C ­ GA ­ U (uracil)­mRNA is synthesized in antiparallel direction

Ex. 3'­ACC­5' of DNA is template for 5'­UGG­3'

­UGG = codon (triplet of mRNA)­(can also mean triplet on nontemplate DNA strand, aka coding strand)

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The triplet code

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The dictionary of genetic code

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AUG ­ start code and methionine­all synthesized polypeptide strands start with methionine, but then an enzyme may break off methionine

stop codes: UAA, UGA, UAG

Ex. 5'AUGCAAGCUUAA3'

methionine­glycine­alanine ­ stop

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Genetic code is universal

­in all organisms tested AUG always means the same thing

­genes can be transcripted and translated even if they are transplanted in other organisms

­some exceptions: ­in some prokaryotes stop codon can be translated into an amino acid­in some unicellular eukaryotes, may have slight variations of genetic code, not the norm

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How is DNA transcribed?1. RNA polymerase binds to a promoter on

the DNA strand­transcription unit = stretch of DNA that is transcribed into RNA

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2. RNA polymerase separates DNA strands and hooks together the RNA nucleotides­assemble in 5' to 3' direction­do not need a primer­in prokaryotes have terminator ­ signals end of

transcription­downstream = direction of transcription­upstream = opposite direction of transcription

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Is this prokaryotic or eukayotic transcription?How can you tell?

Initiation ­ RNA polymerase binds to promoter, DNA strands unwind, RNA synthesis starts

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Elongation:RNA polymerase moves downstream, elongating in 5' to 3' direction, behind DNA reforms double helix

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­Bacteria have one RNA polymerase to synthesize mRNA and other types of RNA (ex. ribosomal RNA)

­eukaryotes have three types of RNA polymerase: I,II,III

­one used for mRNA synthesis is RNA polymerase II­others transcribe RNA molecules not translated into protein

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3. Promoter determines which strand is being used as a template­in prokaryotes RNA polymerase attaches directly to promoter­in eukaryotes, proteins called transcription factors help RNA polymerase bind to the promoter to start transcription

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Initiation of transcription at eukaryotic promoter

­transcription initiation complex = transcription factors + RNA polymerase + promoter

­part of promoter contains a TATA box

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4. RNA polymerase continues to untwist DNA as it moves downstream­10 ­ 20 bases are paired with RNA at a time­adds bases to 3' end­new RNA comes away from DNA template­DNA double helix reforms­transcribes 60 nucleotides per second

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5. one gene can be transcribed by many RNA polymerases, one after the other so many of the same proteins can be made

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6. Termination­ in prokaryotes ­ terminator sequence where polymerase detaches

­ eukaryotes ­ pre­mRNA is cleaved while RNA polymerase II continues to transcribe DNA, have a polyadenylation signal sequence, (AAUAAA),then 10­35 nucleotides downstream, RNA transcript is cut free

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RNA processing in Eukaryotes

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7. pre­mRNA is then modified to mRNA

Added: ­5'cap ­ modified guanine (first 20­40 bases)­3' end ­ poly­A tail formed (added 50­250 adenine nucleotides)

­functions of botha. help mature mRNA out of nucleusb. help protect mRNA from hydrolytic enzymesc. once in cytoplasm, both help ribosomes attach to 5' end

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RNA processing addition of 5' cap and poly­A tail

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8. RNA splicing­are noncoding sections between the coding sections in the pre­

mRNA that have to be removed ­ noncoding or intervening sequences are called introns­exons = coding regions that are eventually expressed as amino

acids (exons "exit" the nucleus and to be translated)

­introns have short nucleotide sequences before and after them called snRNP's­ small nuclear ribonucleoproteins (150 nucleotides long) = signal for RNA splicing

­RNA in snRNP is called small nuclear RNA(snRNA) recognize splice sites

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snRNP's and other proteins make a spliceosome(cuts intron out and binds exons)

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The roles of snRNP's and splicesosome in RNA splicing

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In some organisms RNA splicing can occur with out spliceosome, occurs using ribozymes (RNA molecules that act as enzymes)

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Why are introns and RNA splicing important?­can control gene activity­helps RNA get out of nucleus

­some genes code for two different proteins depending on what exons remain (alternative RNA splicing)­produces greater # of proteins made than # of genes

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Correspondance between exons and protein domains

­domain = structural and functional region of a protein

ex. active site

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Translation­cell interprets mRNA and builds polypeptide

­gets interpreted by tRNA (transfer RNA)­transfers amino acids to ribosome where they are joined together to make a polypeptide­each tRNA translates a specific mRNA­anticodon region of tRNA is complementary to mRNA codon

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Basic concept of translation

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tRNA = RNA strand 80 nucleotides long­has 3D structure

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How tRNA is specific for amino acid

1. Aminoacyl­tRNA synthetase (20) joins amino acid to tRNA

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2. correct match between tRNA and mRNA­only 45 tRNAs­can pair with more than one mRNA­due to relaxed of base pairing rules­U at 5' end of anticodon can pair with A or G in third position = wobble

­explains how codons can differ in 3rd base, not 1st or 2nd ­ look at chart

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Ribosome = two subunits (large and small)

­made of proteins and RNA molecules (rRNA)­made in nucleolus of eukaryotes­large and small subunits only join together when attach to mRNA molecule

*streptomycin and tetracycline are drugs that affect prokaryotes by inactivating ribosomes

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­have a binding site for mRNA and three binding sites for tRNA

­Psite = holds tRNA and the growing polypeptide chain­A site = holds tRNA carrying the next

amino acid for the chain­E site = exit site, where tRNAs leave the ribosome

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Ribosome

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Steps in translation ­ initiation

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elongation

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termination

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several ribosomes can translate the message from one mRNA at the same time (called polyribosomes or polysomes)

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­after production of polypeptide, protein gets folded into 3D structure and post translational modifications happen

­some amino acids may be chemically modified ex. add sugar or phosphate group

­enzymes may remove one or more amino acids from end of chain

­protein could get cut in two pieces (ie.insulin)­two separate chains may come together

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free ribosomes ­ in cytosol make proteins that dissolve and function there

bound ribosomes to ER or nuclear envelope­makes proteins of endomembrane system and ones secreted from cell

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­ribosomes switch from free to bound­signal peptides in the polypeptide chain will signal the ribosome to attach to the ER

­signal recognized by signal recognition particle

­other signal proteins can be used to attach to mitochondria, chloroplasts, etc.

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signal mechanism for targeting proteins to the ER

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Review of RNA types

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prokaryotic and eukaryotic differences

­RNA polymerases are different­eukaryotes depend on transcription factors

­transcription is terminated differently­ribosomes are different­prokaryotes simultaneously transcribe and translate­eukaryotes ­ transcription is segregated from translation­eukaryotes use signals to target proteins to the proper cell organelle

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Coupled transcription and translation in bacteria

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Mutations allow cells to code for many of the proteins synthesizedMutation­changes in genetic material of a cell or viruspoint mutations involve one base pair of a gene

­if it has adverse effect on offspring = disorder­ex. sickle cell anemia

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Sickle Cell Anemia

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Types of point mutations1. Base pair Substitutions =replacement of one nucleotide and its partner with another pair of nucleotides

a. silent mutation if it doesn't change the amino acid­some base pair mutations alter the protein being made ­ can be troublesome

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b. missense mutations ­ altered codon still codes for an amino acid and makes sense, but not right sense

c. nonsense mutation ­a point mutation that changes a codon into a stop codon

­translation gets stopped early­shorter than normal polypeptide­protein is nonfunctional

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Point Mutations

base pair substitutions

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2. insertions and deletionsaddtions or losses of nucleotide pairs­alters reading frame (frameshift mutation)­produce nonfunctional proteins

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Point Mutations

Base pair insertion and deletion

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MutagensSpontaneous mutations = mutations that happen during DNA replication, repair or recombination that lead to base­pair substitutions, insertions or deletions

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mutagens = physical or chemical agents that change DNA to cause mutations

ex. x­rays,UV light, chemicals

­scientists have come up with tests to test chemicals for potential carcinogenic properties(Ames test)

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Revamp of gene definition:

A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule

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Summary of transcription and translation

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