07 - Proteins and Translation

Polypeptides are linear sequences of ____

amino acids

amino acids are linked by _____

peptide bonds

peptide bonds join the _________end of one amino acid to the _________ end of another.

join the carboxyl end of one amino acid to the amino end of another

structure of amino acids

amino end

carboxyl end

side chain (r group)

hydrogen atom

amino terminus is also known as

n- terminus

carboxyl terminus is also known as

c-terminus

Polypeptides:

Linear chains of amino acids linked by peptide bonds

subunits

Individual polypeptide chains in a multi-chain protein; can fold independently and contribute to overall function.

Proteins

One or more folded polypeptide subunits forming a functional 3D structure,

To be functional, a protein must do what

fold into the correct 3-D shape, include the correct cofactors/subunits, and contain any required post-translational modifications

Folding is promoted by

non-covalent interactions

Enzymes involved in the folding process often have

weak affinity for the ribosome and stay near the exit tunnel

Protein domains

the structural units of proteins

usually folds independently and has a particular function

Different proteins can contain the same domain

How could the same DNA-binding domain be found in multiple different proteins?

• exon shuffling by transposition

• gene duplication and divergance

protein domain relation to evolution

Modular and reusable across different proteins

Key to protein function diversity

Products of gene evolution (domain shuffling and duplication)

Different proteins can vary greatly in size and number of polypeptide

___

subunits

1 polypeptide could contain _____ domain

more than 1

1 protein often contains _____ domains with _____ functions

many

different

is the translation of genetic code conserved or no

One of the highly conserved and most complex process in both eukaryotes and prokaryotes

overall process of Translation of the genetic code

1. Initiation - Ribosome attaches to mRNA and begins translating at the initiation/start codon (AUG → methionine) → puts the ribosome in the correct reading frame

2. Elongation – Polypeptide chain elongation → series of steps repeated over and over until a stop codon is reached (UAA, UAG or UGA)

3. Termination – Stop codon signals to stop further elongation and release the polypeptide

Structure of ribosome

Each ribosome has three sites for association with tRNAs:

1. A (aminoacyl) site

2. P (peptidyl) site

3. E (exit) site

Peptide bond formation during elongation

A new peptide bond forms between amino group of incoming amino acid and C-terminus of the growing chain

translation - elongation

Step 1: AA-tRNA binds to an empty A site

Step 2: A new peptide bond is formed between the growing chain and the new amino acid (peptidyl transferase activity)

Step 3: Large subunit translocates

Step 4: Small subunit translocates by 3 nucleotides (codon), resulting in empty A site that can accept another AA-tRNA

The process repeats until the ribosome reaches a stop codon on mRNA

Translation Elongation Factors

are additional proteins that improve the efficiency and accuracy of translation (EF -Tu and EF -G in bacteria)

EF -G

Hydrolyze GTP to drive transitions in the ribosome subunits

accuracy check as Translation Elongation Factors

Accuracy checks:

• Small subunit rRNA hydrogen bonds with the codon -anticodon

• A tight (correct) codon/anti -codon pairing triggers a conformational change in the ribosome and hydrolysis of GTP by EF -Tu (in this image). EF -Tu then releases the AA -tRNA, freeing it for addition of the AA to the growing chain

how is the reading frame set

Beginning from the AUG start codon allows the ribosome to correctly set the reading frame

directionality of ribosome movement

The ribosome moves 5’ to 3’ along the mRNA

directionality of protein synthesis

the polypeptide is built N-terminus to C-terminus (and in prokaryotes, translation can begin at the 5’ end while RNA polymerase is still synthesizing)

The start codon is at the ____ end of the protein-coding sequence in the RNA

5’

AUG codes for

for methionine, so the initial amino acid at the amino (N) terminus of the polypeptide is always Met (but this can be removed later)

initiator factors in eukaryotes and prokaryotes

In bacteria called IFs and in eukaryotes called eIFs.

Translation initiation in prokaryotes

In prokaryotes, the small ribosomal subunit binds to the first AUG codon guided by a specific sequence: The Shine-Dalgarno Sequence (5’-AGGAGG-3’)

Shine-Dalgarno sequence is complementary to a sequence near 3’ end of 16S rRNA → positions the ribosome at the correct spot

Initiation factors are attached to the small subunit

In bacteria the initial AUG methionine is a modified version called N-formylmethionine (fMet)

Initiator tRNA (carrying fMet) interacts with AUG at what will be the ‘P’ site of the ribosome

IFs are then released and large subunit can bind

Shine-Dalgarno sequence

5’-AGGAGG-3’

where is the Shine-Dalgarno sequences located and why is tgis imp

Shine-Dalgarno sequences can be located anywhere along the mRNA

Therefore, prokaryotic ribosomes can synthesize multiple proteins from a single RNA

IF1:

Helps with attachment to mRNA

IF2:

GTP-binding protein that is required for attachment of first AAtRNA

IF3:

Prevents premature attachment of large subunit

Translation Initiation in Eukaryotes

Processed 5’ and 3’ ends are important for translation initiation and for nuclear export • helps ensure only completed mRNA are translated

Eukaryotes have larger ribosomes and require additional proteins (12 eIFs or more) → initiation is more complex than prokaryotes

eIFs bind to the small subunit and are important for:

Initiator tRNA (Met) binding the ‘P’ site with GTP-bound eIF2

Small ribosomal subunit (along with eIFs and initiator tRNA) finds 5’ end of mRNA → scans along until reaches a Kozak sequence (5’ -CCACCAUG C -3’)

GTP bound to eIF2 is hydrolyzed and is released along with other eIFs •

Dissociation of initiation factors allows the large subunit to attach

eIF1s:

Conformational change to allow binding of mRNA

eIF2

Initiator tRNA (Met) binding the ‘P’ site with GTP-bound

eIF3:

interaction with eIF4G on mRNA complex

mRNA has its own set of eIFs:

eIF4E

eIF4A

eIF4G

eIF4E

binds to 5’ cap

eIF4A

has helicase activity that uses ATP hydrolysis to unwind any double stranded regions in mRNA

eIF4G

links 5’ cap and 3’ poly(A) tail. This converts mRNA into a circular message and interacts with eIF3 on small subunit

The 5’ to 3’ scanning activity searches for the what seq in eukaryotes

Kozak sequence

Kozak sequence

(5’-CCACCAUGC-3’)

consensus sequence (we know which nucleotides are found most commonly at each position)

 “leaky scanning” for Kozak sequences

Leaky scanning occurs when the ribosome skips a weak Kozak sequence and initiates at a downstream AUG.

The actual sequence can vary slightly, but the more different it is, the less efficient initiation of translation at that AUG will be

What effect do you think Leaky scanning could have on the proteins produced from an mRNA?

this could create proteins with different amino acid sequences at the n-terminus

Translation Termination

Translation continues until ribosome reaches a stop codon for which no corresponding tRNA is available

Release factors (which resemble tRNA and recognize stop codons) bind in the vacant A site and catalyze the addition of water instead of an amino acid, which frees the C-terminus (no longer attached to a tRNA)

Polyribosomes

Multiple translation initiations typically take place on the same mRNA

Multiple ribosomes associated with an mRNA → Polyribosome (or Polysome)

Attach at AUG, start translating and once AUG is free, another ribosome can assemble

Prokaryotes vs Eukaryotes:Initiation Signal

Prokaryotes - Shine-Dalgarno sequence

Eukaryotes - Kozak sequence (surrounding AUG)

Prokaryotes vs Eukaryotes: Initiator tRNA

Prokaryotes - fMet-tRNA

Eukaryotes - Met-tRNA

Prokaryotes vs Eukaryotes: mRNA Structure

Prokaryotes - Often polycistronic

Eukaryotes - Monocistronic

Prokaryotes vs Eukaryotes: Polysomes

Prokaryotes - Form during transcription

Eukaryotes - Form after mRNA processing/export

Prokaryotes vs Eukaryotes: Initiation Factors

Prokaryotes - IF1, IF2, IF3

Eukaryotes - eIF1–eIF4 (more complex)

Prokaryotes vs Eukaryotes: Ribosome Binding

Prokaryotes - Small subunit binds Shine-Dalgarno via rRNA

Eukaryotes - Small subunit scans from 5′ cap