Polypeptide Synthesis

How does the DNA control the cell’s functions? DNA makes proteins which dictate the function of the cells ONE GENE ONE ENZYME - Beadle and Tatum concluded that each step in the metabolism of arginine is controlled by a separate enzyme and that each enzyme is controlled by a specific gene - therefore a change in the gene would affect the enzyme and therefore affect the metabolism of the individual One Gene One Enzyme meant that each gene was responsible for the production of one enzyme

(All necessary nutrients) (Missing key nutrients)

EXPERIMENT RESULTS Class I Mutants Class II Class III Wild type Minimal medium (MM) (control) MM + Ornithine Citrulline Arginine Working with the mold Neurospora crassa, George Beadle and Edward Tatum had isolated mutants requiring arginine in their growth medium and had shown genetically that these mutants fell into three classes, each defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here, tested both their one gene–one enzyme hypothesis and their postulated arginine pathway. In this experiment, they grew their three classes of mutants under the four different conditions shown in the Results section below. The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements

CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Class I Mutants (mutation in gene A) Class II in gene B) Class III in gene C) Wild type Gene A Gene B Gene C Precursor Ornithine Citrulline Arginine Enzyme A B C

CLARIFICATION not all proteins are enzymes therefore ONE GENE ONE PROTEIN however, not all proteins are made of one polypeptide - some are conglomerates (quaternary structure) of different kinds of amino acid chains - each chain being made by a different gene therefore ONE GENE ONE POLYPEPTIDE

From Gene to Protein Three Basic Steps: 1. Transcription - DNA to RNA - RNA copied from DNA Participants: DNA (Promoter region, TATA box, Transcription Unit, Terminator region), Transcription Factors, RNA polymerase, RNA nucleotides 2. RNA Processing Participants: Pre-RNA (Introns and Exons), SNRNPs, GTP, Adenines 3.Translation: RNA to Protein Participants: m-RNA, t-RNA, amino-acyl t-RNA synthetase, ribosomes, Amino Acids, Release factor

Figure 17.4 Gene 1 Gene 2 Gene 3 DNA strand (template) mRNA Protein molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A C G T U Trp Phe Gly Ser Codon 3 5

TRANSCRIPTION process where one side of the DNA is copied into RNA Template Strand - side of the DNA copied (3’5’) section of DNA transcribed is called the transcription unit Differences in DNA and RNA deoxyribose : ribose Thymine: Uracil 2 strands: 1 strand

Composition of DNA to be Transcribed 1. Promoter region: - TATA Box - Start Point 2. Transcription unit – sequence that actually is copied into RNA 3. Terminator region

Transcription is 3 steps 1. Initiation 2. Elongation 3. Termination

1. INITIATION Process: 1. Protein transcription factors bind to the TATA box in the promoter region – allows RNA polymerase to bind 2. RNA polymerase binds to the start point 3. Additional transcription factors bind and help RNA polymerase to unwind the DNA - Transcription factors (proteins) must attach to the TATA box first -

Polymerase, Transcription Factors and Promoter together are called the TRANSCRIPTION INITIATION PROCESS

Polymerase, Transcription Factors and Promoter together are called the TRANSCRIPTION INITIATION PROCESS

Transcription initiation complex RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A TATA box Start point Template DNA strand 5 3 Transcription factors Promoter RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters 1 Several transcription 2 Additional transcription 3

The attachment of the transcription factors is guided by enhancer sequences of DNA upstream from the transcription unit. Activator proteins bind to the enhancer regions causing the DNA to bend. This guides the transcription factors in place to aid RNA polymerase binding

2. ELONGATION - building of RNA along DNA Template strand (side of DNA copied is determined by promoter) - RNA is built in the 5’ to 3’ direction by adding to the 3’ end of the RNA - RNA polymerase moves along the DNA and builds the RNA COMPLIMENTARY to the DNA sequence (U for T)

Transcribe: GCCATCTAAATCGGG CGGUAGAUUUAGCCC - the DNA closes together after the polymerase has passed until the RNA polymerase reaches the termination point

3. TERMINATION RNA polymerase reaches the terminator segment of DNA and signals for the release of the polymerase from the DNA - in prokaryotes, this ends transcription - in eurkaryotes, the polymerase moves past the terminator point slightly onto an AUAAAsequence and then is released - this allows for the modification of the RNA later

Figure 17.7 Completed RNA transcript Promoter Transcription unit RNA polymerase Start point 5 3 Rewound RNA transcript Completed RNA transcript Unwound DNA Template strand of DNA

Transcription Video

Basic types of RNA 1. Messenger RNA = mRNA 2. Transfer RNA = tRNA (translation) 3. Ribosomal RNA = rRNA (makes ribosomes) 4. Small nuclear RNA = snRNA (for RNA splicing) 5. Signal Recognition Particle RNA = SRP RNA – makes ribosomes bind to rough ER

6. Small nucleolar RNA = sno RNA (aid in ribosome formation) 7. Small interfering RNA = siRNA (gene regulation) 8. MicroRNA = miRNA (gene regulation)

RNA PROCESSING Pre-mRNA must undergo modifications before it can leave the nucleus 1. Modification of the ends 2. Removal of Introns and Splicing of Exons

1: modification of the ends 5’ end - promoter end - capped with a guanine triphosphate (GTP) molecule Two Functions 1. prevents mRNA from degrading 2. attachment site for Translation 3’ end - terminator end - capped with a Poly A tail - long chain of adenines - prevents degradation

A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end Protein-coding segment Polyadenylation signal Poly-A tail 3 UTR Stop codon Start codon 5 Cap 5 UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P 5 3

2: Splicing Eukaryotic DNA is filled with large sections of DNA that are not coded for proteins - copied in transcription - must be removed or it would change the resulting polypeptide sequence  shape  function

Non-coded DNA = intron (“in” for intervening or interrupting) - once thought to be “junk DNA” but now thought to have regulatory functions in gene expression Coded DNA = exon (“ex” for expressed) - only 3% of the human genome codes for proteins – rest is not expressed (introns), codes for the different RNAs or ????

exons spliced together Coding segment TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide 5 Cap Exon Intron 1 5 30 31 104 105 146 3 Poly-A tail Introns cut out and exons spliced together Coding segment 3 UTR

- pre-mRNA must have the introns removed and splice (join) the exons Completed By: small nuclear ribonucleoproteins (snRNP  “snurps”) - snurps - attach to signals at the ends of the introns and, with proteins, form a SPLICEOSOME - cuts out the intron and attaches the ends of the exons

RNA transcript (pre-mRNA) Exon 1 Intron Exon 2 Other proteins Protein snRNA snRNPs Spliceosome components Cut-out intron mRNA 5 1 2 3 Analogy: pre-mRNA: Watching TV with commercials mRNA: Watching TV on DVD

RNA Processing Video

TRANSLATION Basics: 1. m-RNA binds to ribosome 2. t-RNA matches up to m-RNA and brings in AA 3. AA chain built 4. polypeptide detaches

Amino acids tRNA with amino acid attached Trp Phe Gly tRNA Anticodon TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly A G C U Codons 5 3

Background STRUCTURE OF t-RNA: sequence of RNA transcribed from the DNA - 80 nucleotides long - specific three dimensional conformation (foldings and turnings) - "L" shape

3' end to attach to amino acids - series of three nucleotides that match to m-RNA codon - called ANTI-CODON (complimentary to m-RNA)

- matches specifically to the first two nucleotides but the third is not as fixed  WOBBLE effect - can match to one or more bases and therefore GGU and GGC can code for the same amino acid TOTAL: about 45 different t-RNAs - bind specifically to one type of amino acid accomplished by : AMINOACYL-tRNA SYNTHETASE

STRUCTURE OF RIBOSOME - rRNA and proteins 2 subunits: large and small - join together when bound to mRNA Large subunit: 3 binding sites for the building the AA chain A site - aminoacyl tRNA site P site - peptidyl tRNA site E site - exit site

Eukaryotic and Prokaryotic Ribosome differences - eu bigger and slightly different molecularly allows for targeting by antibiotics

TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Exit tunnel Growing polypeptide tRNA molecules E P A Large subunit Small Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. (a) 5 3

TRANSLATION: the details 3 steps: 1. initiation 2. elongation 3. termination

Initiation: Formation of Translation Initiation Complex 1. m-RNA binds to small subunit on "attach here" signal in 5'cap 2. binding of first amino acid = START METHIONINE ALWAYS: AUG codon 3. attachment of large subunit start methionine-tRNA in "P site"

process assisted by protein ‘initiation factors’ CODON (m-RNA) triplets are lined up in the different sites of the ribosome - ready for elongation

Elongation 1. Codon recognition: next matching tRNA comes in with appropriate AA to A site

2.Peptide bond formation: ribosome transfers AA chain from P site tRNA to A site tRNA - forms a peptide bond by dehydration synthesis powered by GTP

3. Translocation: shifting of ribosome complex down mRNA - tRNAs bonded to mRNA and slide down into next site on ribosome A to P P to E - empty tRNA in E site leaves (exits) leaving the A site open and P site has tRNA with elongated AA chain

process continues until Termination

Termination Ribosome reaches a STOP codon: UAA, UAG, UGA - brings a protein called a release factor instead of an amino acid - binds into A site - - no transfer of AA acid chain - hydrolyzes AA chain from tRNA - tRNA exits - Ribosome complex disassembles

Translation Video Translation II

Polyribosomes: once initiator complex is open another ribosome can attach

FINISHED PRODUCTS 1. Cytosolic ribosomes: Finished proteins take on three dimensional shape with the help of chaperone proteins - direction conformation of protein (post-translational modifications)

2. Bound ribosomes: - start as cytosolic ribosomes - at beginning of translation, the mRNA codes for a signal peptide that attaches to SIGNAL RECOGNITION PARTICLE (SRP) - brings ribosome into the ER - Growing polypeptide chain enters the ER cisternal space - secretion proteins enter the whole way - membrane proteins stay bound to the ER membrane

CHANGES IN THE DNA = mutation Chromosomal mutations: changes that affect the whole chromosome Point mutation: change that affects only one nucleotide in the DNA EXAMPLE: Hemoglobin and Sickle cell Hemoglobin In normal DNA code is CTT which is made into RNA (GAA) In Sickle cell the DNA code is CAT - second T is replaced with A - therefore RNA becomes GUA GAA = glutamine GUA = valine - one nucleotide changes one amino acid which changes the shape of the protein which changes the shape of the cell which causes all kinds of health defects

the wild-type template has a T. In the DNA, the mutant template strand has an A where the wild-type template has a T. The mutant mRNA has a U instead of an A in one codon. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Mutant hemoglobin DNA Wild-type hemoglobin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val C T A G U 3 5

TYPES OF POINT MUTATIONS Substitution: one base pair replaces another 1. Silent - WOBBLE effect Ex: CCG changed to CCA Result: GGC changed to GGU - both bring in glycine

Base-pair substitution Wild type A U G C mRNA 5 Protein Met Lys Phe Gly Stop Carboxyl end Amino end 3 Base-pair substitution No effect on amino acid sequence U instead of C Ser Missense A instead of G Nonsense U instead of A

2. Missense: replacement of base pair that brings in another amino acid that changes the structure of the protein Ex: Sickle cell

Base-pair substitution Wild type A U G C mRNA 5 Protein Met Lys Phe Gly Stop Carboxyl end Amino end 3 Base-pair substitution No effect on amino acid sequence U instead of C Ser Missense A instead of G Nonsense U instead of A

3. Non-sense: substitution where change in DNA sequence brings in a stop codon in the middle of the protein - protein never is finished

Base-pair substitution Wild type A U G C mRNA 5 Protein Met Lys Phe Gly Stop Carboxyl end Amino end 3 Base-pair substitution No effect on amino acid sequence U instead of C Ser Missense A instead of G Nonsense U instead of A

Frameshift Mutations 1. Insertion - additional nucleotide is added to DNA shifting the amino acid sequence - changes all amino acids after insertion

2. Deletion - same affect due to loss of nucleotide Frameshifts (both insertion and deletion) are usually most devastating if its one or two nucleotides - insertion or deletion of a triplet may only result in the loss or gain of one amino acid - may or may not disrupt protein