17- gene to protein.pdf

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17- gene to protein.pdf

How does DNA control a cell?

◦ By controlling Protein Synthesis.

◦ Proteins are the link between

genotype and phenotype.


What makes a firefly glow?


Central Dogma of Gene

Expression


Complete the Comparison chart for DNA and RNA


DNA and RNA are both Nucleic Acids

Structure DNA RNA

Sugar

present

Nitrogen

bases

present

Number of

Strands

Relative

length

deoxyribose

ribose

A, C, G, T A, C, G, U

2 1

Long, contains

hundreds or

thousands of

genes

1 3

types one three

Shorter, about the

length of one gene

SIMPLE

SKETCH

mRNA

rRNA

tRNA


The Genetic Code

◦ Sequence of bases in DNA correspond

to the sequence of amino acids in

proteins.

◦ There are only 4 bases but 20 naturally

occurring Amino Acids.

◦ “Codon” = three consecutive bases

code for each amino acid

◦ One gene codes for one protein


Codon Dictionary

◦ Start- AUG (Met)

◦ Stop- UAA

UAG

UGA

◦ 60 codons for the

other 19 AAs.


Dictionary of the genetic code

◦ Each amino acid

except “start” has

multiple codons.

◦ This helps safeguard

against error in

transcription (writing)

or translation

(reading) the code.


Code Redundancy

◦ Third base in a codon shows

"wobble”.

◦ First two bases are the most important

in reading the code and giving the

correct AA.

◦ The third base often doesn’t matter.


Comment

◦ "Wobble" effect allows for 45 types

of tRNA instead of 61.

◦ Reason - in the third position, U can

pair with A or G.

◦ Inosine (I), a modified base in the

third position can pair with U, C, or

A.


Importance of Wobble

◦ Allows for fewer types of tRNA.

◦ Allows some mistakes to code for

the same AA which gives exactly

the same polypeptide.


Reading Frame

◦ The “reading” of the code is every three

bases.

◦ Ex: the red cat ate the rat

◦ Ex: ATT GAT TAC ATT

◦ The “words” only make sense if “read”

in this grouping of three.


Code Evolution

◦ The genetic code is nearly universal.

◦ Ex: CCG = proline (all life)

◦ Reason - The code must have evolved

very early. Life on earth must share a

common ancestor.

◦ Biotech applications use the universal

nature of DNA to move genes from

species to species.


Flow of

genetic info

◦ Transcription - to

write the code to

mRNA

◦ Site of transcription

is the cytosol

(bacteria) or nucleus

◦ Translation - to read

and relate the code

with tRNA and rRNA

◦ Site of translation is

the ribosome


Transcription – the DNA directed

synthesis of RNA

◦ Promoter – specific

nucleotide sequence

along the DNA

◦ In Eukaryotes these

promoters include a

TATA box

◦ Terminator – end of

transcription = UAA,

UAG & UGA


Step by Step through Transcription

◦ INITIATION

◦ RNA polymerase

binds to promoter

◦ This cause the DNA

to unwind & separate

◦ RNA transcript

begins to form


ELONGATION

The template strand is

transcribed by adding

complementary RNA

nucleotides in 5’ 3’

direction

◦ The RNA transcript

grows and DNA

reforms double helix

◦ In Prokaryotes, the

transcript is

immediately usable as

mRNA


TERMINATION

◦ Once a termination

sequence is

transcribed, the

RNA transcript is

released and RNA

polymerase

detaches from DNA

◦ There are three

termination codes


RNA Modification in Eukaryotic cells

◦ 5’ cap and Poly- A tail are added to RNA

transcript

◦ Prevents breakdown of DNA

◦ Facilitates passage through nuclear

pores

◦ Cap provides point of attachment at

ribosome


RNA Processing - RNA Splicing

◦ INTRONS – non-coding segments of

eukaryotic genes

◦ EXONS – coding segments

◦ During RNA processing, introns are

removed and exons are spliced together


RNA Processing - RNA Splicing

◦ Spliceosomes – an

assembly of

snRNPS -

(ribonuclear

proteins) + other

proteins bind to the

end of introns –

recognize code

◦ Newly spliced exon

–only mRNA can

exit nucleus


Functional and Evolutionary

importance of Introns

◦ Introns may play

regulatory roles in the

cell.

◦ Benefit of split genes is

to enable a single gene

to encode more than

one kind of protein.

◦ Facilitates the shuffling

among genes

promoting evolution


Alternative Splicing

◦ The RNA can be spliced into different

mRNA’s.

◦ Each different mRNA produces a

different polypeptide.

◦ Ex. – this explains how the body can

create infinite number of antibodies.


Another Example

◦ Bcl-X L – inhibits apoptosis

◦ Bcl-X S – induces apoptosis

◦ Two different and opposite effects!!


DSCAM Gene

◦ Found in fruit flies

◦ Has 100 potential splicing sites.

◦ Could produce 38,000 different

polypeptides

◦ Many of these polypeptides have been

found


Commentary

◦ About 60% of genes are estimated

to have alternative splicing sites.

◦ One gene does not equal one

polypeptide.

◦ The work of Beadle & Tatum is

being undone with the power of

technology and better

experimentation!


Transcription Review

◦ A biologist inserts a gene from a human

liver cell into a bacterial chromosome. The

bacterium transcribes the gene into mRNA

and translates it immediately into a protein.

The protein produced is useless and is

found to have many extra amino acids than

does the protein made by the eukaryotic

liver cell. Explain Why.


Protein Synthesis

◦ Translation is the

RNA-directed

synthesis of a protein.

◦ one codon one

amino acid

◦ Transfer RNA (tRNA)

molecules interpret

the genetic code as

written on the mRNA

transcript


Role of Transfer RNA

◦ Links mRNA codon

to its matching

amino acid.

◦ “Anticodon” –

complementary

base sequence to

mRNA codon.

◦ Amino Acid

attachment site is

located opposite

the anticodon.


Ribosomes

◦ Structure - Large and

small subunit both

composed of protein

and rRNA.

◦ One enzyme for each

amino acid

◦ Facilitate

• codon – anticodon

complex formation

• peptide bond

formation

◦ Prokaryotic ribosomes

are often the target of

antibiotics


Recognition steps for accurate

translation

◦ 1. Correct match between the tRNA

and the amino acid

◦ Each amino acid has a specific enzyme

that aids in attachment to tRNA –

aminoacyl-tRNA synthetase

◦ 2. Codon-anticodon bonding insures

translation


Initiation of Translation

◦ mRNA binds to a small ribosomal subunit –

AUG is the “start” sequence.

◦ GTP provides energy needed to bring the

large subunit to create a complex.

◦ E = exit ; P = protein building site and A =

enzyme driven Amino Acid binding site.


Elongation of polypeptide chain stop

◦ 1. Codon

recognition

◦ 2. rRNA serves as

a ribozyme –

catalyst of peptide

bond

◦ 3. translocation –

ribosome shifts

the mRNA by one

codon – both

move relative to

eachother


Termination

◦ Stop codon reaches “A” site.

◦ Release factor matches stop codon &

protein is released.

◦ Ribosome complex disintegrates.


Free vs. Bound Ribosomes

◦ Free Ribosomes

• Protein is needed in cytosol

◦ Bound ribosomes

• Protein is needed at membrane or

beyond via ER

• True of cells that line secretory organs

or tissues


Signal recognition particle carries the

complex to the ER membrane

20 amino

acids long

◦ Signal recognition protein temporarily binds

ribosome to the ER membrane

◦ Protein is fed into cisterna as it elongates


Mutations –changes in DNA

◦ Chromosomal mutations - large scale

changes to chromosomes

◦ Point mutations – small scale changes

in just one base pair of a gene

• Substitution

• Insertion

• Deletion

Drosophila antennapedia gene

mutation


Mutations

◦ If Mutation occurs in a gamete or cell

that creates gametes, it may be

transmitted.

◦ If mutation has an adverse affect on

phenotype it is called a genetic disorder

or hereditary disease.


Molecular Basis of Sickle-cell disease

◦ One change in the base sequence leads

to the production of an abnormal

protein.


Base pair substitutions

◦ Silent mutations – no

effect on encoded

protein due to

redundancy

◦ Missense – still codes

for an amino acid but

not the “right sense”.

◦ Nonsense* – a stop

codon in mid gene

* leads to nonfunctional

proteins


Base pair insertion mutations

◦ Addition or loss of base

pairs Disastrous

effect

◦ Frame-shift mutation – #

of nucleotides added or

deleted is not a multiple

of three severe

missense.

◦ why is a multiple of 3

okay?


Frame-shift mutation examples

◦ CAT ATE THE RAT becomes . . .

◦ CAR TAT ETH ERA T

◦ CAR TER ATE THE AT

Disease

related

mutation

statistics


Summary of transcription & translation

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