Wave 9 Chapter 13

Archibald Garrod

Was interested in alkaptonuria, which is the bodies of patients accumulating abnormal levels of homogentric acid (alkapton) which causes black urine and discoloration of cartilage and skin. Was a genetic recessive metabolic defect that Garrod called “inborn error of metabolism”

Beadle and Tatum

They worked on fungus to ask if one gene led to either one enzyme or many enzymes. They obtained nutritional (conditional) mutants that couldn't synthesize methionine (bacteria can synthesize all 20 amino acids but we, humans, cannot) (humans can roughly make 10 amino acids but have to get the other 10). There were many genes required to make one amino acid. They used mutagenesis with light to form the mutants to find what is required to make methionine. They had one type of plate with minimal media which they used because it just had food and would force the fungus to synthesize everything. The other type they could use but didn’t was rich media, which also contained all the amino acids, salts, vitamins, etc. that an organism would need. After mutation, they made 2000 mutant strains, and they put the fungus mutants on to minimal plate with 19 amino acids (excluding methionine) bec some mutants weren’t able to survive on such plate, so they knew which fungi were the mutant they wanted. (one gene one enzyme was what was decided to be the result, but in reality its one gene encodes multiple polypeptides, not one enzyme. furthermore some genes don’t even encode polypeptides).

translation

interpretation of one language to another. in genetics, its the nucleotide language to amino acid language of proteins. Relies on genetic code.

polypeptide denotes…

structure

protein denotes…

function.

codons

groups of 3 nuc. what the genetic info is coded in mRNA as.

Is the Genetic code essentially universal?

Yup

How many codons are there?

3 bases are for one codon, and there are 64 codons because there are 4 bases! 4³ = 64. ( a doublet code would not be able to encode for enough amino acids! Only 16 < 20 aa needed (aa = amino acid)

degenerate/third base wobble/redundancy

third base of codon doesn’t matter as much as the first 2. some codons code for the same amino acid.

The extra 2 amino acids that we don’t have to memorize (for the 22 amino acid table) (we only need to know the 20)

selenocysteine and pyrrolysine. found in speciality enzymes, encoded by UGA and UAG codons respectively. attached by tRNAs that carry them to ribosome.

functional groups on amino acid backbones (everything but R group) at pH 7

amine and carboxylate groups

New amino acids get linked where?

C-terminus

number of peptide bonds = ?

number of amino acids - 1

Is the Genetic Code Overlapping?

No. This means that the 3 codons that are read once at a time are not read again in previous or future phases of translation. Each codon and nitrogenous base in that codon is only read ONCE.;

tRNA are like…

adaptors—> they dock to codons using complementary anticodons and carry amino acids covalently.

Primary structure of proteins

the amino acid sequence.

Folding during translation?

while being translated, further folding will occur as ducted by the amino acid sequence and chaperone proteins.

Secondary structure

protein folds to form regular repeating shapes. Occur due to H-bonds with backbone structure (NOT THE R GROUPS). Backbone is everything but the R groups. Proline cannot form H bond in its backbone so it is an alpha helix breaker!

2 types of secondary structure

Alpha helices (need at least 6 contiguous amino acids to form, looks kinda like DNA, has torsion angles that are all the same which actually creates the geometry we see) and Beta sheets (made up of 2+ beta strands and at least one beta turn [beta turn requires 4 contiguous amino acids to occur] and beta sheets require at least 6 contiguous amino acids to form. Proteins can either be made up of just one type of secondary structure, but most proteins have a mix of the two throughout their structures.

Hydrophobic interactions in protein folding?

main force driving protein folding. End up on the inside of proteins due to trying to avoid the usually polar solvent that proteins are in.

chaperones

proteins that assist another protein’s folding or fix misfoldings.

tertiary structure

short regions of secondary structure that fold into 3D structure. This is the final conformation of proteins that are composed of a single polypeptide. This structure is determined by hydrophobic and ionic interactions as well as H-bonds and van Der walls interactions or any sort of IMF (intermolecular force) BETWEEN R GROUPS.

Quaternary Structure

only for proteins made up of 2+ polypeptides have this. This is when various polypeptides associate with one another to make functional proteins.

Are proteins largely hydrophobic?

YES! This is because most amino acids are non polar and proteins will go into organic phenol layer in extraction! Like dissolves like.

What do the characteristics of a cell depend on?

the proteins it makes!

Why are dimers used in transcription factors?

Double-stranded DNA recognition sites often contain palindromic or inverted repeat sequences; dimeric transcription factors possess two-fold symmetry that allows them to contact both strands across adjacent major grooves simultaneously

Protein function

transport/movement/shape of cell/organization, cell signaling, cell surface recognition, and enzymes.

enzymes

perform chemical modifications, cleavage, and/or synthesis.

Cracking the genetic Code: Nirenberg

goal was to decipher relationship between base composition and particular amino acids. They made mRNAs using polynucleotide phosphatase to stick RNA nucleotides together (it is an enzyme, does not use template, order of nucleotides is random, uses NDPs). They used GDPs and UDPs to control the concentration in percentages to predict the codon possibilities and frequencies. They then used radio labeled amino acids, and did an in vitro translation assay (cell free translation system that uses ribosomes, tRNAs, and other translation factors. They had 20 test tubes, each one having 19 cold (unlabeled) amino acids and 1 different radio labeled amino acid per test tube. They then used radiation to tell which codons code what. After letting translation occur, they threw the contents of test tubes in 15% trichloroacetic acid (TCA) to precipitate polypeptide to measure radioactivity while purifying it from amino acids (otherwise amino acids would give false radiation signal). They then calculated the amount of radio labeled amino acids in the precipitate polypeptides. They then had to account for and helped discover the degeneracy in the code to calculate/predict genetic code due to the resulting frequencies of the radio labeled amino acids in the polypeptides. They could not decipher the whole genetic code in this experiment.

probabilities vs frequencies

probabilities are always between 0 and 1 while frequencies are probabilities x 100%

Cracking the Genetic Code: Khorana

Used copolymers (linking/coupling enzymatically) to take dinucleotides or tetra nucleotides to link them together to form longer piece of DNA, which they then applied same techniques on RNA. Only difference between him and Nirenberg is how they made the short RNAs (2-4 nuc. long). They found that 3 nuc. long RNA could cause ribosome to bind tRNA, so they mixed one triplet in 20 tubes with each different labeled radioactive amino acid, and only the amino acids stuck to ribosome would be retained. This is called triplet binding assay.

Francis Crick in 1950’s: adaptor hypothesis

tRNA has 2 purposes: 1) recognize 3 base codon in mRNA and 2) carry amino acid that is specific for that codon.

anatomy of a tRNA

nucleotide transferase

enzyme that adds to all tRNA’s the nucleotides ACC to 3’ end of of the tRNA (last nucleotide on the 3’ end is always an A then).

tRNA nomenclature

uncharged: no amino acid attached: tRNA^Phe; charged: amino acid covalently attached; Phe.tRNA^Phe. (Phe is the example amino acid name)

aminoacyl-tRNA synthetase

enzymes that attach aa’s covalently to tRNAs at their 3’ A nucleotide (attached at 3’ ribose on that nucleotide). There are 20 types, one for each aa type (at least in E. coli) and they use ATP in their reaction. They involve 3 different molecules (ATP, tRNA, and the amino acid) and the result is charged tRNA. Error rate is less than 1 in every 10,000 bec it has to be highly accurate. charging tRNA with right amino acid requires checking mainly the anticodon (maybe sometimes modified bases too but not as much).

modified bases in anticodo, can you have them?

Yes! In the third wobble position. Francis Crick proposed in his 1966 Wobble hypothesis that the first 2 positions pari strictly across to A-U and G-C rule, but the third position can tolerate some mismatches.

isoacceptor tRNAs

tRNAs that can recognize the same codon.