Protein Synthesis Notes

Protein

Synthesis:

Overview• Transcription:

synthesis of mRNA

under the direction of

DNA.

• Translation: actual

synthesis of a

polypeptide under the

direction of mRNA.

Transcription

• Process of making RNA from a DNA template.

Transcription Steps

1. RNA Polymerase

Binding

2. Initiation

3. Elongation

4. Termination

RNA

Polymerase:

• Enzyme for

building RNA

from RNA

nucleotides.

• Prokaryotes - 1

type

• Eukaroyotes- 3

types

RNA Polymerase Binding:• Requires that the enzyme find the “proper”

place on the DNA to attach and start

transcription – the Promoter Region.

RNA

Polymerase

Binding Needs:

• Promoter Regions

on the DNA.

• Transcription

Factors.

Promoters

• Regions of DNA where RNA Polymerases

can bind.

• About 100 nucleotides long. Include

initiation site and recognition areas for

RNA Polymerase.

Promoter region at the front of

the gene to be transcribed.

TATA Box

• Short segment of T,A,T,A repeated.

• Located 25 nucleotides upstream for

the initiation site.

• Recognition site for transcription

factors to bind to the DNA.

Transcription Factors

• Proteins that bind to DNA

before RNA Polymerase.

• Each factor recognizes a

different area, such as the TATA

box.

• They each bind to area to “flag”

the spot for RNA Polymerase.

Transcription Initiation

Complex• The complete assembly of

transcription factors and RNA

Polymerase bound to the promoter

area of the DNA to be transcribed.

Transcription Complex• Only when all transcription factors have

been picked up by and bonded to the RNA

Polymerase, can transcription begin.

Initiation

• Actual unwinding of DNA to

start RNA transcription.

• Requires Initiation Factors.

• Getting Transcription started is

complicated.

• Gives many ways to control which

genes are decoded and which

proteins are synthesized.

Elongation

• RNA Polymerase untwists DNA

1 turn at a time.

• Exposes 10 DNA bases for

pairing with RNA nucleotides.

Elongation

• Enzyme builds 5’ 3’.

• That means it transcribes the 3 >

5’ strand – the is called the anti-

sense strand.

• Rate is about 60 nucleotides per

second.

Termination

• DNA sequence that tells RNA

Polymerase to stop.

• Ex: AATAAA

• RNA Polymerase detaches from

DNA after closing the helix.

At the End of Transcription:

• We have Pre-mRNA

• This is a “raw” RNA that will need

processing (or Modification).

Modifications of RNA1. 5’ Cap

2. Poly-A Tail

3. Splicing

5' Cap• Modified Guanine nucleotide added to the

5' end.

• Protects mRNA from digestive enzymes.

• Recognition sign for ribosome

attachment.

• This mRNA will be threaded through a

ribosome like film through a projector.

• The 5’ cap protects the leading edge of

the mRNA from wear and tear.

Poly-A Tail

• 150-200 Adenine nucleotides added to the

3' tail

• Protects mRNA from digestive enzymes.

• Aids in mRNA transport from nucleus.

RNA Splicing

• Removal of non-protein coding regions

of RNA.

• Coding regions are then spliced back

together.

Introns

• Intervening sequences.

• Removed from RNA.

Exons

• Expressed sequences of

RNA.

• Translated into AAs.

Result

Introns - Function

• Left-over DNA (?)

• Way to lengthen genetic message.

• Old virus inserts (?)

• Way to create new proteins.

• Help reduce likelihood of accidental

damaging mutation.

mRNA modification• 1) 5’ cap: modified guanine; protection; recognition site for

ribosomes

• 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport

• 3) RNA splicing: exons (expressed sequences) kept,introns

(intervening sequences) spliced out; spliceosome

Transcription Movie:

Translation

• Process by which a cell interprets a

genetic message and builds a

polypeptide.

Materials Required

• tRNA

• Ribosomes

• mRNA

Transfer RNA = tRNA

• Made by transcription.

• About 80 nucleotides long.

• Carries AA for polypeptide synthesis.

Structure of tRNA• Has double stranded regions and 3 loops.

• AA attachment site at the 3' end.

• 1 loop serves as the Anticodon.

Anticodon• Region of tRNA that

base pairs to mRNA

codon.

• Usually is a

compliment to the

mRNA bases, so

reads the same as the

DNA codon.

Example

• DNA - GAC

• mRNA - CUG

• tRNA anticodon - GAC

Ribosomes

• Two subunits made in the nucleolus.

• Made of rRNA (60%)and protein (40%).

• rRNA is the most abundant type of RNA

in a cell.

Both subunits

Large Subunit• Has 3 sites for tRNA.

• P site: Peptidyl-tRNA site -carries the growing polypeptide chain.

• A site: Aminoacyl-tRNA site -holds the tRNA carrying the next AA to be added.

• E site: Exit site

Translation Steps

1. Initiation

2. Elongation

3. Termination

Initiation

• Brings together:

• mRNA

• A tRNA carrying the 1st AA

• 2 subunits of the ribosome

Initiation Steps:

1. Small subunit binds to the

mRNA.

2. Initiator tRNA (Met, AUG)

binds to mRNA.

3. Large subunit binds to

mRNA. Initiator tRNA is in the P-

site

Initiation

• Requires other proteins called

"Initiation Factors”.

• GTP used as energy source.

Elongation Steps:1. Codon Recognition

2. Peptide Bond Formation

3. Translocation

Codon Recognition

• tRNA anticodon matched to mRNA

codon in the A site.

Peptide Bond Formation

• A peptide bond is formed between

the new AA and the polypeptide

chain in the P-site.

• Bond formation is by rRNA acting as

a ribozyme

After bond formation

• The polypeptide is now transferred from

the tRNA in the P-site to the tRNA in the

A-site.

Translocation

• tRNA in P-site is released.

• Ribosome advances 1 codon, 5’ 3’.

• tRNA in A-site is now in the P-site.

• Process repeats with the next codon.

• Elongation takes 60 milliseconds for

each AA added.

Termination

• Triggered by stop codons.

• Release factor binds in the A-site

instead of a tRNA.

• H2O is added instead of AA, freeing

the polypeptide.

• Ribosome separates.

Translation

Protein Structure

Size and

Shape

Compariso

n of

Proteins

Levels of Protein

Structure2o

3o 4o

1o

Amino Acids

Peptide Bonds• Proteins are formed by creating peptide

bonds between individual amino acids.

– Remove water

– Called dehydration

Peptide Bonds

20

Amino

Acids

Some Amino Acids

have/are:• A Negative Charge

• A Positive Charge

• Uncharged & Polar

• Nonpolar

Amino Acids

HydrophobicHydrophilic

Secondary (2°)

Structure

• Folding into α-

helix or β-

sheets

α-Helix

Myoglobin

β-sheet

β - Sheets

Parallel Antiparallel

2 kinds:

• Parallel

• Antiparallel

β - Sheets

COXSACKIE VIRUS

AND ADENOVIRUS RECEPTOR

Antiparallel

Often both structures are

found in the same molecule:

Tertiary (3°)

Structure

3° Structure

• 3-D Conformation of Protein

• contain “domains” (~50-350

aa) that fold and function

independently

• may contain many domains

Domain 1

Domain 2

Domain 3

Domain 4

Quarternary (4°)

Structure

4° Structure• association of

polypeptides into multi-

subunit protein

Catalase Quaternary Structure

Protein Functions

• Structural

• Regulatory

• Enzyme

• Transport