Protein Synthesis“the job of a gene”

IB Biochemical Biology

I. OverviewA. Explain

DNA (anti-sense strand)

“unzipped” mRNA tRNA Polypeptide

TAC ATACCCACT

AUGUAUGGGUGA

UAC

AUA

CCC

ACU

TRANS-CRIPTION

TRANSLA-TION

CODON ANTI-CODON

Met

Tyr

Gly

STOP

Peptidelinkage

The Genetic Code

B. Practice

DNA (anti-sense) mRNA tRNA Polypeptide

CATAAAGAAACT

B. Practice

DNA (anti-sense) mRNA tRNA Polypeptide

CATAAAGAAACT

GUAUUUCUUUGA

CAUAAAGAAACU

VAL

PHE

LEU

STOP

Definitions…• CODON – triplet of bases on

an mRNA strand 3.5.3

• RNA polymerase – forms an mRNA strand complementary to the ANTI-SENSE strand of DNA (the strand acting as the template) 3.5.2

C. Degeneracy – there is more than one codon for many amino acids Ex: GGU & GGA both code for glycine

D. Universality – all living organisms use the same “triplet codes”

E. One Gene, One Polypeptide-One gene codes for one polypeptide chain-Ex: 2 genes code for Hb

(one for the each of the two alpha-chains, and one for each of the twobeta chains)

There are, of course, many exceptions.

F. Compare DNA & RNA (3.5.1)– Do a chart

Sugar, base, strand number.

II. Transcription... DNA makes mRNA (3.5.2)

A. Transcription terms: in eukaryotes

• RNA Polymerase

1. Splits DNA into 2 strands

2. Rewinds DNA after transcription

3. Uses RNA nucleoside triphosphates

to build mRNA

R Base R Base

Hi-energybonds

NUCLEOSIDE

TRI-PHOSPHATE

NUCLEOTIDE& ENERGY (used to join nucleotides

together to make mRNA)

• Sense strand – has same base sequence as mRNA (except U for T)

• Anti-Sense strand – has same base sequence as tRNA (template strand)– Is the strand TRANSCRIBED into mRNA

• Promoter region – a base sequence causing RNA polymerase to bind & begin transcription (has TATA box)

• Terminator – a base sequence which stops transcription

GATGAAAT

CTACTTTA

sense strand anti-sense

strand

5’ 3’ 5’ 3’

PRO-MOTER(TATA)

TERMI-NATOR

GATGAAAT

CTACTTTA

GAUG

5’

GATGAAAT

CTACTTTA

GAUGAAU

Pre-mRNA strand has been transcribed

(builds in 3’ direction)

(7.3.1, 7.3.2, 7.3.3)

B. RNA splicing (7.3.4, 7.1.5)

pre-mRNA (RNA which was just transcribed)

Intervening sequence (non-coding)Expressed mRNA – will be coded into amino acids

RNA transcript is “cut” by spliceosomes to release the introns (regulatory function but not expressed into proteins), and the exons are spliced together...

• 5’ end capped with a GUANINE NUCLEOTIDE– Functions as an “ATTACH HERE!” sign for ribosomes

• 3’ end given a poly(A) tail… 100+ (A) nucleotidesmature mRNA:

Exon Intron Exon Intron Exon

Exon Exon ExonG AAAAAA

G AAAAAA

III. Translation (3.5.4, 7.4.2)

A. Processed mRNA moves out of the nucleus to the ribosome

B. Structure of the ribosome is 60% rRNA & 40% protein

LARGE SUBUNIT

two tRNA binding sites

P AmRNA binding site

SMALL SUBUNIT

rRNA – ribosomal RNA, made in the nucleolus

C. tRNA activation (7.4.1)1. tRNA structure

a. base sequence ACC at 3’ terminal for amino acid attachment

b. two other major loops

c. triplet of bases (anti-codon) in loop of seven nucleotides

d. sections which become double stranded by base pairing

e. each tRNA has a distinctive 3-dimensional shape

f. There are tRNA activating enzymes that match up amino acids with tRNA

2. tRNA Activating Enzymes (IN CYTOPLASM)

“tRNA- activating” enzyme (one for each amino acid)

Energy from ATP is used to join tRNA to the amino acid... Catalyzed by the enzyme.

tRNA

ATP AMP + 2P + ENERGY

amino acid

ACC

3’

5’

Anti-codon

D. The process of translation

• Many ribosomes move along the same mRNA translating at the same time.

1. Initiation:a) tRNA with anticodon complementary to the mRNA start codon (AUG- methionine) binds to the small subunit of the ribosome

POLYSOME (7.4.3, 7.4.6)

b) Large subunit joins small subunit

c) This initiator tRNA is in P-site

d) Another tRNA with its amino acid arrives and binds in the A-site

e) Energy is supplied by GTP (guanine triphosphate) to form a peptide bond between the two amino acids

2. Elongationa) amino acids are added one by one to

the growing chain

b) tRNA in the A-site is translated to the P-site, taking the mRNA with it

c) ribosome moves 5’3’ on the mRNA (7.4.4)

d) Large subunit moves first, followed by small subunit, chain is ELONGATING

3. Termination

a) ribosome reaches a STOP CODE on the mRNA (ex: UGA)

b) released polypeptide has started folding

c) tRNA detaches, ribosome subunits separate

E. Types of Ribosomes

Free... make proteins used in cytoplasm of the cell

(ex: hemoglobin)

Bound to ER... make proteins secreted by the cell

(ex: digestive enzymes likepepsin)

IV. Gene Mutation– Permanent change in DNA– Affects the protein synthesized

A. Insertion – the addition of a base

DNA: T A A T A A T C C

mRNA:

AA:

DNA: T A A T T A A T C C

mRNA:

AA:

RESULT: “total frame shift” non-functional protein

ex: NO MELANIN = ALBINO

B. Deletion – also gives “frame shift”

C. Substitution – less severe

-no frame shift

-imperfect protein… ex: Sickle Cell

G A G (glutamic acid)

mutated

G T G (valine)

V. Regulation of Gene Expression

• At any particular time, some genes are expressed and making proteins and some are not

• Each gene is regulated separately in eukaryotes (…and in groups called “operons” in prokaryotes)

ONE “OPERON”: a single promoter serves multiple genes; a “transcription unit”

• “Lac operon in E. coli”

1. Regulator gene – codes for repressor protein (this gene is constantly expressed at a low rate, ensuring a continuous supply of repressors)

2. Without lactose, the repressor is active (and bound to the operator)… RNA polymerase cannot move past the promoter

Promoter (TATA) operator GENE #1 GENE #2 TerminatorRegulator gene

3. The repressor is allosteric, and when lactose is present, it binds to the repressor and changes its shape (causing it to fall off of the operator)

4. RNA polymerase can now move past the promoter, and the protein is made (which break down lactose as an energy source for the bacteria)

5. When all of the lactose is broken down, there isn’t any left to fit the repressor’s allosteric site (so the repressor goes back to the operator)

Two types of operon control…(reminder: in bacteria only!)

1. INDUCIBLE – The repressor blocks transcription unless it is stimulated by the presence of a molecule

– Ex: lac operon

2. REPRESSIBLE – The repressor allows transcription unless it is activated to bind to the operator

– Ex: trp operon

• In eukaryotic cells – Many ways to regulate gene expression

1. The organization of chromatin-DNA can be de-activated before transcription occurs (by histone acetylation- adding an acetyl group … –COCH3, or by methylation - the adding of a methyl group… CH3)

2. Transcription-If “transcription factors” are not present to find and bind to the TATA box, mRNA will never be produced

3. Processing of mRNA-If the proper spliceosomes are not present, mRNA will never leave the nucleus

4. Translation (Silencing by RNA interference)-Translational regulation – unique types of RNA can bind to the mRNA, making it impossible for translation to occur

5. Modification of the protein at the endoplasmic reticulum or golgi-addition of carbohydrate chains to proteins…-if the protein is to be short-lived, ubiquitin is added (to mark it for degradation)

VI. The Molecular Biology of Cancer…

• Cancer arises from mutations in genes that regulate cell growth and division– Can be spontaneous, from chemical

carcinogens, physical mutagens (like X-rays), or viruses

– Can be from changes in genes that normally INHIBIT cell division (called “tumor suppressor genes”)

– Can be from changes in genes that allow NORMAL cell growth & division (proto-oncogenes)

p53

The p53 gene is expressed when there has been damage to a cell’s DNA…

gene p53 can make a TRANSCRIPTION protein which suppresses cell (tumor) growth…

REGULATION OF TRANSCRIPTION

TUMOR SUPPRESSOR PROTEIN Made by p53

gene

Acts on DNA to synthesize

GROWTH-INHIBITING PROTEINS

Works as a Transcriptional Factor

Role of p53: 1) slows cell cycles to allow repairs to occur, 2) turns on genes that do repairs, 3) initiates apoptosis (programmed cell death) if damage can’t be repaired

What would happen if a mutation due to environmental factors knocks out the p53 gene?

• Excessive cell growth and cancer!