Gene to Protein

I. What do genes do?

A. Garrod’s work

B. Beadle and Tatum

A. Garrod-Alkaptonuria-19021. inborn error of metabolism2. “unit factor” makes functional ferment

(A)3. recessive (a) makes nonfunctional

ferment4. AA normal Aa makes sufficient5. aa homozygote makes no ferment6. His work proved the link between

genetics and the production of proteins

B. Beadle and Tatum

Beadle and Tatum protocol

II. Protein synthesis-an overview

A. Information flow

• 1. DNA->RNA->protein• 2. the process involves two distinct classes of

molecules• 3. nucleic acids with the monomers of the four bases

A, G, T, C• 4. the proteins with their monomers of amino acids• 5. each possess polymers that differ from other

polymers by the sequence of the monomers• 6. Think of it in a language sense

• 7. DNA->RNA is called transcription• a. a scribe was an ancient who copied original texts• b. DNA serves as the template from which mRNA is

manufactured• c. the language is the same-different dialect• d. will see that the RNA has a different nucleotide

base• e. uracil replaces thymine in the structure of RNA• f. thymine possesses a methyl group that uracil

lacks

• 8. RNA->Protein is called translation• a. in this process you are changing

languages• b. different script as well as vocabularly• c. the order of the nucleotide sequence is

being used to direct the line up of the amino acids in the polypeptide chain that will grow

• d. the site of this translation are the ribosomes of the cell

B. An analogy might help

1. Valuable cookbook2. Copy recipe3. Carry recipe copy to kitchen4. Produce pasta fagiole

• A polypeptide 140 amino acids long would be coded for by a mRNA ________ nucleotides long.

III. The genetic code

A. How long is a genetic word?• 1. codon• 2. the code is redundant but not

ambiguous

B. Code is cracked in 1961 by Marshall Nirenberg

C. Nature of the code

• 1. the code redundant• 2. the code is not ambiguous• 3. one codon never represents

more than one unique amino acid• 4. there are punctuation codons in

the list• 5. the start codon is always AUG• 6. there are also termination

codons

D. How is the code read?

• 1. nonoverlapping or overlapping?• 2. a reading frame is established by the

presence of the start codon• 3. from there on out, the nucleotides are read

every three• 4. Thecatanddogranoutthe• 5. as long as this string is read in the correct

reading frame there is a message• 6. get out of the frame and it is just gibberish

E. Genetic code evolved very early in life• 1. the genetic code is shared by all living forms of life• 2. Wobble theory• a. notice that in the genetic dictionary-most of the

specificity seems to reside in the first two letters of the codon

• b. the third nucleotide of the codon seems to be less important in determining which amino acid is being specified

• c. when there is redundancy it resides in the location of the third base pair

• d. believed that ancestrally there were sixteen amino acids

• e. if there are only sixteen amino acids-codon needs to be only two nucleotides long 42 = 16 unique

combinations• f. it is believed that the third position served as a

spacer• g. with evolution need to expand into the third spacer

position

IV. RNA

• A. Differences between DNA and RNA

• 1. DNA is double helix

• 2. RNA is a single stranded

molecule with sugar phosphate

• 3. uracil replaces thymine

• 4. RNA is a shorter molecule

• 5. RNA can travel from the nucleus to the

cytoplasm

B. Types of RNA-basically three

• 1. messengerRNA (mRNA) is a copy of the gene

• 2. ribosomalRNA (rRNA) produces a portion of the ribosome

• 3. transferRNA (tRNA)

C. RNA production-Transcription

1. Initiation-beginning • a. RNA polymerase is the enzyme of

transcription • b. it attaches to the promoter of the gene

in question• f. almost universal in eukaryotic

promoters is the TATA box• g. found upstream from the start point• h. a transcription factor binds to the

TATA segment before RNA polymerase• i. transcription factors are proteins that

regulate the transcription of the gene• j. they can either turn on or turn off

transcription by regulating accessibility to the promoter by the RNA polymerase

2. Elongation stagesa. RNA polymerase unwinds the DNA double helixb. exposes 20 DNA bases at a timec. serves as a platform for RNA-DNA nucleotide base pairingd. can also only attach in the 5’->3’ directione. produces the chain at the rate of 60 nucleotides/secf. the RNA detaches from the RNA polymerase while the DNA goes

back into helixg. multiple RNA polymerases can ride along the DNA transcribing

multiple copies of the gene in question

3. Chain terminationa. when the RNA polymerase

reaches the termination sequence it releases both the mRNA and the DNA template

b. the release point on eukaryotic mRNA is downstream from the actual termination segment

c. the cleavage point is also the site for the attachment of the polyAAA tail

D. Production of the eukaryotic mRNA produces a primary transcript1. this primary transcript must be

modified before it leaves the nucleus

2. RNA splicinga. an average protein is about 400 amino acidsb. therefore theoretically about 1200 nucleotides are needed for codingc. eukaryotic genes are usually much longer than

thisd. exonse. introns

Some interesting facts about introns

• a. almost nonexistent in the prokaryotic world• b. their frequency seems to increase in the more complicated

organisms• c. sponges have fewer introns than do flatworms• d. flatworms have fewer introns than do round worms• e. as you climb the evolutionary tree-introns become more

ubiquitous• f. in eukaryotic genes, there can be tens to hundreds of

introns which are hundreds of nucleotides long• g. is it fair to call introns junk DNA based on the above?

Some possible functions of introns

• a. safe zones for mutations• b. at a later time in the future the intron could become a functional exon• c. introns may regulate the transcription of the gene• d. splicing of different exons increases protein product• e. introns may represent past viral infections that are in a dormant state

3. Modification of ends

a. 5’ end of the molecule• a. first segment produced• b. receives a cap of GTP• c. seems to serve several functions• d. protects the end first produced from degradation• e. serves as a signal for the ribosome that this is where you

attach• f. might serve as an energy source for the attachment of the

ribosomeb. the 3’ end of the mRNA receives a polyA tail• a. several functions• b. again protection from degradation• c. somehow signals for the export of the mRNA to the

cytoplasm• d. might be used as a counter serving to control the life span

of the mRNA

Completed mRNA transcript ready for translation

V. The synthesis of proteins-translationA. Overview

V. The synthesis of proteins-translation

• A. Overview• 1. all of the amino acids

needed to produce proteins are stockpiled in the cytoplasm

• 2. these amino acids are either synthesized by the body or the amino acids are absorbed from the diet

• 3. a recipe arrives in the cytoplasm containing the order that the amino acids should be assembled in

• 4. the order is present in the sequence of codons possessed on the mRNA

• 6. a ribosome attaches to the mRNA, runs down the mRNA, links amino acids together according to the instructions

•

• 7. key to the process is that there are specific tRNA’s that are unique for each amino acid

• 8. the tRNA’s loaded with a specific passenger are floating around the cytoplasm

• 9. by random molecular collisions exposed codons on the surface of the ribosome attach to corresponding anticodons on the surface of the tRNA

• 10. peptide bonds can be formed between the two adjacent amino acids

• 11. the ribosome can progress along the mRNA to the next codon

B. tRNA’s1. produced in the nucleus like all RNA2. only about 80 nucleotides long3. Internal complementary base pairing4. it folds into a structure that looks a bit like a cloverleaf5. two very important regions of the tRNA6. the anticodon sticks out of one side and the 3’ end has a specific site for the

attachment of an amino acid

C. Aminoacyl-tRNA synthetases• 1. this process occurs in the

cytoplasm of the cell• 2. specificity between each

amino acid and the tRNA that delivers it to the site of protein synthesis

• 3. this depends upon the appearance of specific enzymes that do a job

• 4. aminoacyl-tRNA synthetase• 5. there are twenty different

varieties of this enzyme-each specific in its shape

• 6. the synthetase attaches the amino acid to the tRNA that

is then free to wander around until it joins a ribosome

• 7. who makes the aminoacyl-tRNA synthetases?

D. Ribosomes

1. composed of two subunits-• 2. the ribosomal subunits only

are found together while they are assembled on a mRNA

• 3. the total weight of a ribosome is about 60% RNA and 40% protein

• 4. the rRNA is transcribed in the nuclear region known as the nucleolous

• 5. literally thousands of ribosomes in a cell, rRNA is by far the most common RNA possessed by a cell

• 6. the ribosomes are assembled in the nucleolus from rRNA and proteins imported from the cytoplasm

• 7. the completed subunits are then exported through the nuclear pores and distributed throughout the cytoplasm by the ER

•

• 8. Notice the three sites on the ribosome:• a. P• b. A• c. E• 9. The ribosomes of prokaryotes and

eukaryotes are a bit different• a. function in much of the same way• b. structures are a little different as are

their chemistries• c. antibiotics make use of this subtle

difference in the manner in which they work

• d. some antibiotics can block the translation of bacterial genes while not affecting the eukaryotic cell

• 10. the two parts of the ribosome• a. the smaller subunit keeps the

mRNA in a linear sequence allowing docking of the tRNA’s

• b. it also moves along the mRNA like a train moving along tracks

• c. the larger subunit appears to be the part of the molecule which helps the peptide

bond to form• 11. polyribosome

E. Building a polypeptide-three simple steps

• 1. chain initiation 2. elongation 3. termination

1. Chain initiation

• a. small subunit of ribosome attaches upstream to the start codon

• b. initiator tRNA with methionine recognizes the start codon AUG

• c. through the use of GTP, the large subunit then attaches to finish the formation of the translation initiation complex

• d. notice that the A site on the ribosome is open allowing the next step of translation to begin

2. Chain elongation-grows by 10 amino acids per second

3. Chain termination

4. Polyribosome

F. Signal polypeptides-proteins for export

VI. Mutations-the ultimate source of variation-will only talk about point mutations

A. Substitution-one nucleotide is substituted for a second

1. silent mutation due to redundancy of the code

2. missense mutationa. One amino acid is exchanged

for a second

b. Sickle cell anemia is an example

Change in primary structure

Heterozygote advantage

3. Nonsense mutation-premature termination of translation

B. Insertion-deletion mutations cause a frame shift

C. Which is the worse?