Protein Synthesis

What is the “one gene–one enzyme” hypothesis and what experiment supported it?

Beadle & Tatum showed that mutations in specific genes caused defects in specific enzymes

• Organism: Neurospora crassa (bread mold)

• Used mutagens to create mutants

• Prototroph (wild type): grows on minimal medium

• Auxotroph (mutant): requires added nutrients (e.g., arginine)

• Found that defects in a gene = defects in specific enzymes in the arginine biosynthesis pathway

• Established that genes encode enzymes

What is the modern correction to the “one gene–one enzyme” hypothesis?

One gene → one polypeptide.

• Enzymes are proteins, but not all proteins are enzymes

• Many proteins have multiple subunits encoded by separate genes

• Thus: one gene specifies one polypeptide chain, not necessarily an entire enzyme

• secondly, some genes will encode an RNA molecule that is never made into a protein

• some genes will control what dna sequences are expressed so you will never see their effect outside the nucleus

What are the structural features of RNA?

Single-stranded nucleic acid with ribose sugar and bases A, G, C, U.

• Sugar: ribose

• Nitrogenous bases: A, G, C, U (no thymine!)

• Usually single stranded

• Can fold into complex shapes (important for tRNA, rRNA)

What are the three major types of RNA and their functions?

mRNA, tRNA, rRNA.

1) mRNA (messenger RNA)- this is made during transcription , made from the DNA (in the nucleus), it then leaves the nucleus and carries the message to the cytosol and it has the recipe for building the protein​

​

2) tRNA (transfer RNA)- used during translation and it will specify which amino acid is added to your protein sequence , trna is always folded in this weird structure bc it carries in amino acids​

​

3) rRNA (ribosomal RNA)- ribosomal rna is scattered throughout our genome to ensure that we are always able to make ribsomomes just in case we get a mututaion​

How do RNA viruses and retroviruses replicate their genomes?

RNA viruses use RNA→RNA replication; retroviruses use reverse transcriptase for RNA→DNA.

• RNA viruses (flu, polio): replicate via RNA-dependent RNA polymerase

• Retroviruses (HIV, HPV):

  • Use enyzme reverse transcriptase

  • Convert RNA → DNA

  • Integrate into host genome

What is transcription?

The synthesis of RNA from a DNA template, happens AFTER replication.

ingredients:

-dna template (gene of interest), pair the dna template with complementary rna which is made during this process,

-substrates : ribonucleoside triphosphates, rna polyermase ​

What are the three stages of transcription?

Initiation, elongation, termination.

1) Initation: THIS begins at the promoter sequence and that promoter sequence is a special sequence of dna that rna polymerase can bind to the promoter, what happens is that you unwind the dna strand which makes your promoter available ​

2. Elongation: the dna sequence will be unwound about 20bp at a time, ur mrna sequence will elongate from the 5’ to 3’ primed ends so it is antiparrelel and complementary to the dna template ​

3. Termination:

   • Polymerase encounters termination sequence

   • RNA transcript released

What is a promoter?

A DNA sequence where RNA polymerase binds to begin transcription.

• Defines start site and direction

• In eukaryotes, requires transcription factors

• RNA polymerase recognizes promoter signals to begin RNA synthesis

Every gene needs to have a promoter if it is going to be transcribed, at least one promoter will exist but sometimes there is more than one promoter (it depends on gene)

What major modifications occur in eukaryotic pre-mRNA?

1) Splicing which is when introns removed (not expressed RNA) occurs, next exons (expressed RNA) are ligased

2) Add a bunch of G caps (aka guanines) to the beginning end of mRNA strand, allows mRNA to bind to ribosome

3) Poly-A tail added to 3’ end of mRNA for stability so that the mRNA can leave the nucleus

** only happens for eukaryotes bc pre-mRNA needs to mature b4 it can leave the nucleus

What is the difference between introns and exons?

Introns are removed; exons remain and are expressed.

• Introns: non-coding, removed by splicing

• Exons: coding sequences that remain

• Mutations affecting splicing can cause disease

What is the spliceosome and what does it do?

A large RNA–protein complex that removes introns.

• Made of snRNPs (small nuclear ribonucleoproteins)

• Recognizes intron boundaries

• Catalyzes cutting and ligating exons together

• Involves RNA catalysis (ribozyme activity)

What is alternative splicing?

Producing different mRNAs from the same gene by rearranging exons.

• Major source of protein diversity

• Allows one gene → multiple proteins

• Regulated by splicing factors

• Example: Tropomyosin gene has multiple variants

in the situation with pre mRNA, we can do alternative splicing where we combine different exons for expression (ex: putting exon 1 and 2 together or instead putting exon 2 and.3 together) ​

​

What is a codon?

A triplet of mRNA bases that codes for an amino acid.

• 64 codons total

• 61 code for amino acids

• 3 stop codons: UAA, UAG, UGA

• Start codon: AUG (methionine)

What are key features of the genetic code?

Nearly universal, redundant, non-overlapping.

• Universal: shared by almost all organisms

• Redundant: multiple codons per amino acid

• Not ambiguous: each codon = single amino acid

• Non-overlapping, no punctuation

​

- there are many amino acids that are encoded for by more than one codon (which makes sense bc there are 20 amino acids and 60 codons that can make amino acid), each codon can only encode a specific amino acid… in other words each codon will only incorportate the same amino

What are key features of tRNA?

Cloverleaf structure with anticodon and amino acid binding site.

• Amino acid attaches to 3’ end

• Anticodon base-pairs with codon

• Folding stabilized by hydrogen bonds

• Structure essential for accurate translation

each tRNA has the region that is known as the anti-codon , the anti-codon is complementary to the codon and the anti-codon determines which amino acid the tRNA carries , 64 different codons and therefore they all have anti-codons that will ​

​

How are amino acids attached to tRNA (charging)?

Aminoacyl-tRNA synthetase attaches the correct amino acid to its tRNA.

• Requires ATP

• Ensures amino acid and anticodon match

• One synthetase per amino acid

• Critical for translation accuracy

What is the wobble effect?

Flexibility in the 3rd base of codon–anticodon pairing

Allows fewer tRNAs to recognize more codons

• Helps speed translation

• Only affects 3rd base; first two bases are strict

What are the major components of a ribosome?

ribsomomes are the site of protein synthesis/ aka site of translation​

- they have both a small and large subunit and they only come together for translation otherwise they are hanging out seperatley ​

​

Large Subunit:​

- will have multiple sites : T site, A site, P site, E site​

• At the A site the tRNA anti-codon binds complementary to the mRNA codon ​

• P site, the tRNA will add the amino acid to the growing polypeptide chain (actually making a peptide bond there) ​

• E site, (think exit site) , the uncharged tRNA will arrive and then leave the ribosome ​

• Small Subunit – will validate that the 3 base pairs between mRNA and tRNA are correctly matched , and does so by checking for hydrogen bonds, it is the wrong tRNA then the wrong tRNA will be ejected and it will just wait for the correct tRNA to arrive ​

What happens during translation initiation?

Initiation complex forms at start codon “AUG”

Components:

• Small ribosomal subunit

• mRNA

• Initiator tRNA-Met

• Initiation factors

• Large ribosomal subunit

• Complex assembles upstream of UTR sequence

• Start codon always codes for methionine

beings translation, and we need to make the initationcomplex, the initation complex is made of the small subunit, largesubunit, mRNA and tRNA, this is going to bind upstream of ursequence , the initaton factor is an enzyme that aids this process ,the tRNA that always joins is always the methymine tRNAspecifically at the start codon AUG​

What occurs during elongation in translation?

Amino acids are added one by one to the growing polypeptide.

• Charged tRNA enters A site

• tRNA in P site transfers chain → tRNA in A site

• Catalyzed by peptidyl transferase

• Ribosome shifts (translocation)

• Empty tRNA exits through E site

• Requires elongation factors

during elongation, we form that polypeptide. This willhappen is that the tRNA that has methumine will pass themethymine to the newly arrived tRNA that is in the A site , the newtRNA slides from the A site to the P site, during this processpeptotrysthase will be used to create a peptide bond btwn the aminoacids , now the polypeptide chain will be in the P site (associatedwith that tRNA) and the one that left its amino acid (the empty tRNA)is in the E site and then it leaves; the enzyme that is creating thebonds , another enxyme that helps this process is the elongationfactor ​

What triggers termination of translation?

Encountering a stop codon.

• Stop codons: UAA, UAG, UGA

• Release factor enters A site

• Polypeptide released

• Ribosomal subunits detach

What is a polysome?

Multiple ribosomes translating the same mRNA simultaneously.

• Increases efficiency

• Common in both prokaryotes and eukaryotes

What determines where a newly made protein will go?

Signal sequences and targeting mechanisms.

• Cytoplasmic proteins: may use chaperonins

• ER-bound proteins:

  • Have hydrophobic leader sequence

  • Bound by signal recognition particle (SRP)

  • Inserted into ER during translation

-Protein can be translated and then released into the cytoplasm, in this case, the protein generally stays in the cell but often on your protein you can have a called a localization signal which tells the protein where to go (think a little address protein like similar to on a letter) this will tell the protein if it needs to go to a certain organelle or if it can just stay in the cytoplasm ​

​

​

-in the process of entering the organelle, you have this localization signal, once you get to the organelle it binds to a docking protein (helps the protein interact) , next there are also chaperonin proteins will help the protein enter into the organelle and will also help fold the protein properly ​

-another thing that can happen with proteins is if proteins are destined to go to the endoplasmic reticulum, translation will stall and now it will relocate , once it attaches to the endoplasmic reticulum (so taking the whole complex , stalling it, and relocating to er) ; once it reaches the er , once the ribsome reasscoaties, there will be a singaling sequence (aka leader sequence) it will interact with a singla recognition particle and then interacts with the receptor protein on the er membrane , the polypeptide will begin to be fed into the receptor , this will then allow for translation to continue , once in the er the signal sequence is removed, as translation continues the polypeptide is fed into that er lumen , once translation hits the stop codon and is terminated that protein ends up in the endoplasmic reticulum , once in the er they can be modified (ex adding sugars) ​

How does a signal sequence direct proteins into the ER?

SRP binds signal peptide and escorts ribosome to ER.

• SRP pauses translation

• SRP receptor docks ribosome to ER membrane

• Protein threads into ER lumen

• Signal often cleaved off

What are examples of post-translational protein modifications?

after you’ve made the polypeptide you are gping to do a couple of other things to it including:​

​

Proteolysis- cleave (chop it off or remove portions ) the protein in order to activate it , ex in blood clotting proteins need to be cleaved to activate them​

Glycosylation- add sugars ​

Phosphorylation- adding phosphate group to change the shape of protein which would either activate or deactivate it ​

How do many antibiotics inhibit translation?

us and bacteria have the same genetic code, however, humans have different ribsomomes then bacteria do so a good way to attack bacteria cells with antibiotics is to tharget their ribosomes , inhibit their protein synthesis and synthesis of their cells walls, which is a great way to kill them and leave us unharmed​

Ex: Diphtheria targets eukaryotic translation​

Translation

happens after transcription is complete

-for prokaryotes, translation can begin b4 transcription is complete bc there is no nucleus

-for eukaryotes, your transcript has to be modified before it can leave the nucleus and then translation can begin​

What happens once the singal sequence directs a protein into the er?

once in the er they can be modified (ex adding sugars) , from the rough er you send the proteins in vesicles to the golgi apparartus which processes and packs up the proteins and put them into vesicles and we then send these proteins out of the cell, the golgi also makes lysosomes so sometimes these proteins will also go to making lysosomes . ​

​