Topic 6-L2- Translation Flashcards
Proteins are _________. They are comprised of polymers of amino
acids connected by __________
polypeptides. peptide bonds.
Amino acids have an
amino group on one side, an ⍺–carbon in the middle (has “R group”) and a carboxyl group on the other side.
Peptide bonds are between the
carboxylic acid group of one amino
acid and the amino group of the next amino acid.
proteins are directional based on how
polymers are assembled
from N-terminus (free amino terminus) to C-terminus (free carboxylic acid terminus
There are 20 different amino acids that make up proteins. They have the
same backbone, but different R groups.
primary structure
The chain (sequence) of amino acids in a protein
secondary structure.
Small segments of protein adopt simple local structures (local in 3D space, not necessarily in sequence)
Most common secondary structure elements are
⍺-helices & β-sheets. Formed by hydrogen bonding in peptide backbone (amide H & carbonyl O)
Tertiary structure.
The full 3D structure of a protein
Will typically include multiple secondary structure elements arranged in different ways & other structural features as well
Quaternary structure is the result of
multiple different polypeptides
coming together – multimeric proteins (or protein complexes).
The individual polypeptide chains in a multimeric protein are
subunits. Can be:
- identical (homomeric)
- different (heteromeric)
Proteins contain “domains” - these are
structural and/or functional
segments. Can be small or large - most proteins contain a few (or more)
different domains
A given domain is defined as having a particular
structure and/or carrying out a particular function. Typically, a given domain will be found in a number of different proteins.
Common domain example
“helix-turn-helix” (HTH) domains
HTH domains bind DNA – found in
> 200 different proteins in any given Salmonella genome…mostly DNA-binding regulatory binding proteins.
The ribosome uses tRNAs to
convert (“translate”) the mRNA sequence into a protein sequence
Each tRNA has a specific
anticodon that binds a particular three base codon.
At other end, tRNAs carry the specific
amino acid (cognate amino acid) that corresponds to that codon.
tRNA synthetases are the enzymes that
“charge” tRNAs – add the amino
acid to the CCA at the 3’ end.
“wobble” –
same tRNA for 2 different codons
start codons – mostly
AUG, GUG, UUG.
In E. coli:
83% AUG, 14% GUG, 3% UUG.
In bacteria, start codon is translated to
N-formylmethionine (fMet)
(chemically modified version of methionine) using a special tRNA.
AUG encountered during normal translation encodes a standard
methionine. fMet often removed from proteins after translation.
In Archaea/Eukarya, __________ is used
methionine (unmodified)
The three stop codons are
UAA , UGA & UAG
Prokaryotic ribosome is the 70S ribosome – is made up of two subunits
– 30S (small subunit) and 50S (large subunit). Each subunit is comprised of rRNA and ribosomal proteins.
30S & 50S interact dynamically
associate/dissociate during translation
E. coli has ribosomes like
- 30S ribosome containing 16S rRNA And 21 proteins.
- 50S containing 5S/23S rRNA and 31 proteins.
rRNA carries out much of the main function of ribosome – including
catalyzing peptide bond formation.
For each ORF, bacterial mRNAs contain a
ribosome binding site (RBS) also known as a Shine-Dalgarno sequences (with 5’ UTR).
The RBS binds to
16S rRNA in a free 30S ribosomal subunit (no 50S) – helps ribosome locate bona fide start (typically AUG) codons.
Free 30S subunit binds RBS, fMet tRNA binds AUG. Using GTP for
energy, 50S subunit recruited –
_________ forms.
full 70S ribosome
The ribosome has 3 tRNA binding sites:
- A (aminoacyl) site
- P (peptidyl) site
- E (exit) site
A (aminoacyl) site:
Where new charged tRNAs enter and recognize the codon being translated. Growing peptide from P site is transferred to the amino acid carried by the tRNA. Translocation occurs – RNA moves 3 bases (one codon).
P (peptidyl) site:
Second position - tRNA moves to P site. Now this tRNA transfers growing amino acid chain to new charged tRNA that has entered the A site. Once complete, tRNA lacks amino acids (uncharged)
E (exit) site:
Uncharged tRNAs exit here
During translation, going through the three RBS repeats until ribosome encounters a stop codon. Once this occurs, a protein called a
release factor binds – releases peptide and mRNA – 30S/50S dissociate. Ribosome free to begin again.
The same mRNA can be simultaneously translated by multiple different ribosomes – multiple ribosomes on a single transcript called
polysomes
transcription and translation are often
coupled in prokaryotes – RNA being translated while transcription still going on (RNA polymerase and ribosomes both attached to transcript)
Each eukaryotic mRNA encodes
one gene – contrasted prokaryotic
transcripts encode 2+ ORFs (ribosomes re-initiate on same transcript)
Main mechanistic differences in the initiation step. 5’ cap recognized
by
initiation complex in eukaryotes (ribosome binding site). More regulation/complexity at this step.
Eukaryotic ribosomes are larger and a bit more complex then prokaryotes -
40S (small subunit)
60S (large subunit)
80S complete ribosome
Chaperones are proteins that help
other proteins adopt their properly
folded and fully active state
Do all three domains use chaperons?
Yes
Chaperones functions:
initial folding, re-folding denatured proteins, helping subunits in multimeric proteins come together, preventing aggregation, incorporating cofactors into enzymes…there are even RNA chaperones
How do chaperones gain energy?
ATP hydrolysis
Special chaperones are activated in response to
high or low temperatures (heat shock / cold shock proteins) to assist with protein/RNA folding
In E. coli major chaperones include
DnaJ/DnaK, GroEL and GroES.
NarJ is a different sort of chaperone. It inserts an
essential cofactor – Moco (contains molybdenum) - in the enzyme nitrate reductase
All proteins are synthesized by
ribosomes in the cytoplasm
All prokaryotes use
translocase systems that transport proteins across (and into) the cytoplasmic membrane.
Most translocated proteins contain a signal sequence at the
N-terminus that targets the protein to a particular secretion system – often removed after translocation.
The Sec secretion system & twin arginine translocase (Tat) are
ubiquitous in prokaryotes – others are more specialized
The Sec secretion system recognizes a signal sequence in the first ~20
amino acids of protein -
translocates unfolded protein before it folds
Protein either passed across
- cytoplasmic membrane (SecA pathway)
Or
- recognized by RNA/protein complex – signal recognition particle - and inserted into membrane (SRP pathway).
Both pathways pass the unfolded protein through a membrane channel - Sec YEG translocon
Both pathways for transporting proteins require
ATP
Tat pathway
secretes folded proteins – proteins that
must fold in cytoplasm
