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CHAPTER 17

FROM GENE TO PROTEIN

One Gene – One Enzyme Hypothesis

� Archibald Garrod – 1st to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions ; alkaptonuria

� Beadle & Tatum – bread mold, Neurosporacrassa; isolated mutants strains requiring arginine; concluded that each strain was defective in a single gene (see textbook for full explanation of experiment)

Revisions: One Gene – One polypeptide Hypothesis

� Not all proteins are enzymes (keratin, insulin)

� Some proteins constructed of more than one polypeptide chain (hemoglobin)

Basic Principles of Transcription & Translation

� Transcription – synthesis of RNA under the direction of DNA (serves as a template); occurs in nucleus of eukaryotic cells

� mRNA – messenger RNA; carries the genetic message from DNA to the ribosome

� Codon- sequence of mRNA nucleotides that code for a specific amino acid

� Translation – synthesis of polypeptide; cell translates base sequence of mRNA into amino acid sequence ; occurs in ribosome

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Transcription

� RNA polymerase – separates DNA double helix and adds RNA nucleotides in the 5’ to 3’ direction; does not need a primer � RNA polymerase I,II,III

� RNA pol II – one used to for mRNA sequences that are translated into proteins

� specific sequences of nucleotides mark where transcription can begin – promoter

� Promoter serves as the binding site for RNA polymerase & which DNA strand serves as the template

Stages of Transcription

� 1. Binding & Initiation

� Group of proteins plus RNA polymerase II bind to the promoter region of DNA forming the initiation complex

� Promoter commonly contains a TATA box – nucleotide

sequence about 25 nucleotides upstream from the start of transcription

� 2. Elongation

� RNA polymerase moves along the DNA, continuing to untwist the double helix

� RNA nucleotides added to 3’ end of growing chain

� As complex moves down the DNA, the double helix re-

forms, while new RNA molecule pulls away from the DNA

template

� 3. Termination

� Polyadenylation signal sequence (AAUAAA) is formed

� Polymerase continues transcribing past this sequence (100’s

of nucleotides)

� Polymerase eventually falls off

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Modification of mRNA

� 5’cap and Poly A tail – added to pre mRNA – protect against degradation in cytoplasm

� Introns – noncoding regions interspersed within the coding regions

� Exons – coding regions

� Introns are spliced out and exons are joined together

� Splicing is done by small nuclear ribonucleoproteins(snRNPs)

� snRNPs join together to form operating called spliceosomes.

Translation

� Message is a series of codons along the mRNA, the interpreter is the transfer RNA or tRNA

� tRNA transfer amino acids from the cytoplasm to the ribosome

� Cell cytoplasm stocked with all 20 amino acids� tRNA has an amino acid at one end and a specific sequence of nucleotides at the other called the anticodon

� Aminoacyl-tRNA synthetase – enzyme that binds the correct amino acid to the tRNA

Ribosomes

� Divided into 2 subunits – large & small� Composed of proteins & ribosomal RNA rRNA� Made in nucleolus of eukaryotes� Structure aids the bringing together of mRNA codon & tRNA anticodon

� Ribosome has binding site for mRNA� Also binding sites for tRNA

� P site (peptidyl-tRNA site) – holds tRNA carrying growing polypeptide chain

� A site (aminoacyl-tRNA site) – holds the tRNA carrying the next amino acid to be added

� E site (exit site) – discharges the tRNA from ribosome

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Building a Polypeptide

� 3 steps – initiation, elongation & termination

� Requires protein “factors” & energy (hydrolysis of GTP – guanosine triphosphate – similar to ATP)

Initiation

1. Small ribosomal subunit binds to mRNA searching for AUG

2. A tRNA having the anticodon UAC (initiator tRNA) pairs to the start codon AUG – carries the amino acid methionine

3. Attachment of the large ribosomal subunit occurs� Proteins called initiator factors bring all components

together

� Initiator tRNA is in P site

� Vacant A site ready for next amino acid

� Elongation – a.a. added one by one ; involves the participation of proteins called elongation factors ; 3 step cycle

1. Aminoacyl-tRNA base pairs with the mRNA codon in the A site ; requires energy from hydrolysis of GTP

2. RNA molecule of large subunit catalyzes the formation of a peptide bond (between carboxyl of one a.a. & amine of another)

3. Translocation – ribosome translocates the tRNA in the A site to the P site ; emptying tRNA in P site is moved to the E site then removed ; mRNA moves along with its bound tRNA’s,bringing the next codon to be translated into the A site

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� Termination

1. When a ribosome reaches a stop codon (UAA, UAG,or UGA) on mRNA, the A site of the ribosome accepts a protein called release factor instead of tRNA

2. Release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain; polypeptide is freed from the ribosome

3. Two ribosomal subunits & all other components dissociate

Protein Synthesis Summary

Point Mutations & Protein Structure & Function

� Mutations – changes in genetic information of a cell

� Point mutations – chemical changes in just one

base pair of a gene

� If in gamete, it can be transmitted to offspring &

future generations

Types of Point Mutations

� Base – Pair Substitution – replaces one nucleotide & its complementary partner with another nucleotide� Silent mutations – due to redundancy of genetic code,

these have no effect on the protein (CCG mutated to CCA still codes for glycine)

� Missense mutations – altered codon still codes for an amino acid & makes sense but not the right sense

� Nonsense mutations – substitution inserts a stop codon prematurely resulting a polypeptide that is too short

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Types of Point Mutations

� Insertions & Deletions

� Additions or losses of nucleotide pairs

� More serious effects on proteins

� Frameshift mutation – occurs when an insertion or deletion

causes the improper grouping of nucleotides in a codon

� http://learn.genetics.utah.edu/units/basics/transcribe/

� http://207.207.4.198/pub/flash/26/transmenu_s.swf

� http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html

� http://library.thinkquest.org/20465/g_DNATranscription.html