RNA translation

Genetic code

encodes all 20 amino acids

multiple codons for most amino acids

Start codon

AUG start codon

What are the start codons

UAA

UAG

Reading frames…

Defines the amino acid sequence

We define it by looking for the AUG (methionine) sequence and then start grouping the RNA into 3s

Silent mutation

nucleotide pair substitution that doesn’t change nucleotide

Missense mutation

nucleotide base-pair substitution

amino acid changesN

Nonsense mutation

Nucleotide bp substitution

causes a stop codon (UAA or UAG) to appear

Nucleotide-pair deletion can result in

Frameshifts..

What can frameshifts result in

can cause immediate missense (amino acid change) or nonsense (stop codon) both, or none!

None happens when a whole amino acid is added or a whole amino acid (3 bps) is deleted

tRNA (4 points)

The link between mRNA and protein sequence


1. recognizes codon on the mRNA and brings in the proper amino acid
2. 80 nucleotides long
3. transcribed from genes as per usual
4. **Base-pairs with itself to form cloverleaf shape**

* amino acid is binded to tis 3’ end
* anticodon, which basepairs with codon antiparallel and complenetary

How long are tRNAs?

80 nucleotides long

Anticodon

* anticodon, which basepairs with codon on the mRNA antiparallel and complenetary

Dihydrouridine

‘DD’ on left loop

A base that got modified after transcription

Pseudoeuridine

Ψ

A base that got modified after transcription

Are there Us in DNA?

Yes, sometimes due to mutations

Are there only Gs As Us and Cs in RNA?

No, you have pseudouridine and dihydrouridine that changed after transcription

How codon redundancy is managed for translation

More than one tRNA (different anticodon for different codons of the same amino acid)

but some tRNA have recognize multiple codons due to the **wobble position**

Wobble position

5’ on the anticodon, 3’ on the codon. 3RD POSITION OVERALL.

U can have either A, G, or I.

I is inosine, another modified base

Each base on the codon can have different possible anticodon bases

EUKARYOTES ARE LESS WOBBLY THAN PROKARYOTES

2 sequential steps in ensuring fidelity

→ aminoacyl-tRNA synthetases

→ base pairing

aminoacyl-tRNA synthetases

Proteins that recognize the tRNA and put on the proper amino acid using ATP reaction.

**APPROXIMATELY** 20 synthetases, **1 FOR EACH** AMINO ACID, depends on # of amino acid since not all bacteria have 20

Capable of error correction by **hydrolytic editing**

Hydrolytic editing

If aminoacyl-tRNA synthetases happen to put the wrong amino acid on the tRNA they can backspace/delete it

Recognition of a specific tRNA by its synthetase

* identifies (since it has to bind to) anticodon nucleotides
* recognizes the nucleotide sequence of the 3’ acceptor stem/arm (where amino acid is added)
* reading nucleotide sequences at additional positions on the tRNA

Ribosomal structure

* Made up of different proteins and RNA molecules
* large subunit and small subunit
* 3 important sites, E site P site and A site

Where are ribosomes located

* on endoplasmic reticulum (rough?)
* in cytosol

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A site P site and E site and what order they appear in

A site = aminoacyl site

* The next tRNA w next amino acid added

P site = peptidyl site

* tRNA with growing polypeptide change

E site = exit site

* where tRNA leaves

Overview of translation

* energy stored in covalent bond btwn amino acid and tRNA in P site makes peptide synthesis energetically favourable (moving peptide bond from P site tRNA to A site tRNA)
* peptidyl transferase activity of the rRNA in the large subunit

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What catalyzes peptide bond formation?

peptidyl transferase activity of the rRNA in the large subunit

Explain a ribozyme

RNA molecules that posess catalytic activity

* therefore, the ribosome is a ribozyme, because **A, P, E sites are primarily made up of RNA molecules**

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Does the ribosome have a backspace mechanism? How does elongation and quality control happen

Elongation Factors

* EF-Tu in **prokaryotes** (**EF1 in eukaryotes**) checks aminoacyl tRNA
* Ef-G (prokaryotes) (EF2 in eukaryotes)

EF-Tu/EF1

* binds to GTP
* ribose and 3 phosphates
* It checks base-paring. if base paring is not correct, EF-Tu (or EF1) is not released, peptide bond can’t form
* **if base-pairing is correct** GTP is hydrolyzed and EF-Tu/EF1 is released. tRNA is then in the right position, there is a slight delay for one last check before p.p bond formation

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EF-G/EF2

helps the ribosome to move the mRNA forward one codon and helps speed up elongation of the polypeptide chain

* basically moves the small subunit which “moves” the mRNA

Can ribosomes perofrm protein synthesis without the aid of elongation factors?

Yes, but slower and inefficient

Role of elongation factors (2)

improve speed and efficiency

error checking function

How are elongation factors mediated?

GTP hydrolysis (breaking it apart to form GDP and Pi) and release of EF-Tu & EF-G

mRNA structure bacteria vs. Eukaryotes

Bacteria:

* ribosome-binding sites and non-coding sequences
* you can have multiple proteins from one mRNA, due to having multiple coding sequences (polycistronic)
* coding sequences, meaning multiple starts and stops

Eukaryotes:

* Monocystronic (codes for one protein) doesn’t have multiple ribosomal binding sites?
* One singular coding sequence creates one protein
* also has 2 protein sequences
* **has a cap and poly-A tail**

PROKARYOTES: Order of initiation of translation (4 steps)

1. Shine-Dalgarno sequences on mRNA base pair with rRNA in small ribosomal subunits
2. Positioning of small subunits to initiating AUG codons on mRNA also requires Initiation Factors (IFs)
3. fMethionine aminoacyl tRNA binds to initiator codon (AUG)
4. Large ribosomal subunit binds

Shine-Dalgarno Sequences

sequences that allow for binding to rRNA in small ribosomal subunits

EUKARYOTES: Initiation of translation steps

eIF4E (Eukaryotic initiation factor) binds to 5’ cap

eIF4G binds to eIF4E and poly-A binding proteins which is binded to poly-A tail

* Eukaryotic mRNA MUST have a loop for extra quality control check


1. Small ribosomal subunit with translation IFs and INTIATOR TRNA ALREADY BOUND without RNA

* Finds AUG met start codon
* Then large ribosomal subunit comes in

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Termination of translation

* Specific protein translation release factor recognizes stop codon, mimics tRNA structure to fit into A-site (both eukaryotes and prokaryotes)

Polyribosomes (eukaryotes or prokaryotes?)

**Both euk. and prok.**

A bunch of ribosomes on an mRNA

Relatively slow protein synthesis

one ribosome every \~80 nucleotides

the spiral shape

Protein folding (chaperone proteins)

Proteins that help other proteins fold

exaples of molecular chaperones:

* Hsp60
* Hsp70

Hsp stands for heat shock protein

* when heat shock causes protein to denature, these proteins help the protein fold back up

Post-translational modifications (list them and why they may be required)

Many proteins require post-translation modifications such as:

* phosphorylation
* glycosylation

Covalent modifications may be required to:

* make protein active
* recruit protein to correct membrane or organelle

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Control of protein degradation

Some proteins are short-lived, while others may last for months or years.

**Proteins targeted for degradation** have a small protein called **ubiquitin** covalently attached to them, (creating a polyubiquitin chain) which **directs them to the proteasome** where they are **degraded by proteases**, and the **amino acids recycled into new proteins in the cell, ubiquitin is recycled as well, not destroyed**

Antibiotics (what they are) and modes of action (3 points total)

1. Antibiotics: act on **prokaryotes**
2. **Not all,** but many inhibit **translation**


1. acting on **ribosome**
3. **Drugs**

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Things that act on both bacteria and eukaryotes or just eukaryotes are basically just poisons for us

Describe information flow. Transcription to translation, the whole shebang.