Term 2 Lecture 1: Translation Flashcards
Proteins serve a number of biological functions
-light produced by fireflies is a luciferin+ATP reaction catalysed by luciferase enzyme
- fibroin proteins make up spider web
- castor beans contain the toxic protein ricin
Translation of an mRNA molecule takes place on a ribosome.
Translation is the process by which mRNA is read by a ribosome to form a protein.
Ribosomes are protein synthesising factories each made up of one large and one small subunit. Size of ribosomes differs between pro and eukaryotes. Both small and large RNA units are a mix of RNA and proteins.
Many relatively small proteins per subunit. RNA predominates the subunit structure
Ribosomes 2 subunits
Large subunit and small subunit
30S and 50S in bacteria
=70S
40S and 60S in eukaryotes
= 80S
S is a nonlinear measurement hence they don’t add up
Ribosome composition
Eukaryotes 80S
large subunit
60S
28 SrRNA (4718n)
5.8 SrRNA(160n)
55 rRNA (120n)
- 50 proteins
Small subunit
40S
185 rRNA (1874n)
- 33 proteins
Bacteria 70S
Large subunit
50S
23SrRNA(2904n)
5 SrRNA (120n)
-34 proteins
Small subunit
30S
165 rRNA (1541n)
-21 proteins
n = nucleotides here
Eukaryotes have 3 RNA’s in their large subunit whilst bacteria have 2. Both eukaryotes and bacteria small subunits have only one RNA.
Many proteins are incorporated too
One bacterium can have 2000 ribosomes they make up 10% of cellular protein and 80% of cellular RNA
How ribosomes are named (S)
Cell extract is layered ontop of a sugar gradient in a test tube (most conc at bottom of tube) the tube is then centrifuged at 500,000g for several hours.
Cell components form bands depending on their sedimentation coefficients
S stands for Svedberg units. Heavier molecules or ribosomes sink quicker and further giving them a higher unit number.
Sizes are measured in Svedberg units after the Swedish biochemist Theodor Svedberg
Svedberg or S units are nonlinear. They measure the rate at which a particle sediments (moves to bottom) in a liquid when subjected to high centrifugal force
Rate depends on size, density and shape - hence why it is nonlinear
1 Svedberg unit = 1 micrometer per second when subjected to acceleration of 1 million X gravity
Ribosomes are ribozymes
Active site of ribosome is peptidyl transferase, activity is conferred not by a protein as in most enzymes but by an rRNA (there are no proteins in the active site.) A ribosomes active site is at the interface between the two subunits where rRNA is, the protein part is on the outward facing sides
2 important non-coding RNA’s
rRNA- ribosomal RNA, forms part of the large and small ribosomal subunits
tRNA- transfer RNA, brings correct amino acids to the ribosome to be added to the new protein
Ribosomal RNA (rRNA) structure
It folds into a complex secondary structure crucial for rRNA to catalyse the peptide bond.
rRNA is a single continuous molecule but forms multiple structures/domains. Stable secondary structure allows accessory proteins to attach to the ‘scaffold’ form.
Structure of E. Coli 16 SrRNA was discovered by experiments and computation. As well as usual base pairing (G-C, A-U) non standard base pairs can also form (e.g. G-U) in double stranded regions of the RNA molecule - usually at the edge of a non-base paired region
Transfer RNA (tRNA) function and structure
Function
Brings aas to the active site of the ribosome. Aas are selected to be added to growing polypeptide chains in an order specified by the sequence of mRNA. Each AA is brought to the chain by a tRNA specific to that particular aa.
Structure
See diagram start of notebook 2
tRNA has a site called the acceptor arm that recognises and attaches to a specific AA and a site called the anticodon loop that recognises the matching codon on the mRNA.
tRNA was hypothesised to exist by Crick many years before it’s actual identification.
tRNA is the adaptor molecule that can attach to specific aas and read the mRNA code.
Often described as having a clover structure
2 crucial regions
1) anticodon - complimentary to codon on mRNA this part reads the code. 3’ end always ends CCA, terminal adenine is covalently linked to the specific AA. Specificity comes from shape and modifications to tRNA. This allows the tRNA to be recognised by it’s specific amino acyl tRNA synthetase.
Over 50 different modifications to tRNA are known. Most act to improve specificity/accuracy of the tRNA/ AA recognition. One of the most common is methylation of the base sugar component of the nucleotide
2) the acceptor arm - binds to an AA. This arm is formed by the binding together of 7bp of the 5’ and 3’ end of the molecule. The AA is attached to the adenosine on the 3’ end.
-The D loop (or arm) has a modified di-hydrouridine base.
-T loop (or arm) named after the sequence thymidine-psuedocridine (another modified uridine base)
-The extra loop aka variable loop may only be a few bp or much longer.
Aminoacyl tRNA synthetases join a specific AA to the tRNA
Carboxyl group (COO-) of AA attached to hydroxyl group of 2’ or 3’ carbon atom of the first nucleotide at the 3’ end of the tRNA where adenine always is.
Specific Aminoacyl-tRNA’s (tRNA+AA) assembled for each different AA.
AA+ ATP →pyrophosphate (2Pi)+AA AMP
Then adding tRNA to aa AMP causes the formation of Aminoacyl tRNA and AMP is released.
Aminoacyl tRNA synthetases catalyse this 2 step reaction, highly specific, one enzyme for each AA (20 in total)
In the first step an activated as intermediate is produced that has a link between its carboxyl group and AMP. In the process ATP is cleaved producing the energy for the next reaction to join the AA to tRNA.
The AA AMP remains bound to the enzyme whilst the second stage occurs.
Here tRNA takes the place of AMP forming Aminoacyl tRNA so the AA becomes a passenger of tRNA.
In this way each tRNA forms a covalent link with its specific AA by aminoacylation (aka charging)
AA is attached to the acceptor arm, covalently linked between COO- in the AA and 3’ OH group in the terminal nucleotide of tRNA (that is always adenine)
Aminoacyl tRNA synthetase is accurate and corrects its own mistakes
Error rate of synthesis is 1 in 1000 (10³)
This 2 stage action and the editing site in the AA tRNA synthetase give high accuracy.
tRNA synthetases remove their own coupling errors through hydrolytic editing of incorrectly attached aas. The incorrect AA us rejected by the editing site
The initiator tRNA
First codon is always AUG and specified Met
Special initiator tRNA is needed for the first AA different to the one required for subsequent Met addition ( this is because initially there are no other AAs to bind to)
Eukaryotes use Met-tRNAi ^met (i for initiator)
Bacteria use Met-tRNA^fmet and their first methionine is N formyl methionine, the formyl group is usually removed later (often with the whole methionine)
Whatever the organism, first AA (methionine) is brought by initiator tRNA (special tRNA only for the first AA.)
In bacteria ONLY this initiator has a formyl (-HCO) group attached to the N of the amino group (hence called N-formyl methionine) this is a different tRNA to the one used for met residues that are internal to a protein.
In most proteins the formyl group is removed often with the methionine attached after synthesis of approximately the first 20 aa of the peptide chain - it’s unclear why it is needed.
In modification after translation proteins lose methionine (and other AAs) hence many mature proteins when characterised (purified) do not have N-terminal methionine - but all of them start with one!
Translation phase 1: initiation - assembly of small subunit (prokaryotes)
See diagram start of notebook 2
In all proteins the first AA is always met, in bacteria it is formyl-met.
In bacteria translation initiation complex is built up directly at the initiation codon, where protein synthesis begins (in eukaryotes a more indirect process to locate the initiation point - large and small subunits remain in the cytoplasm until they need to assemble)
In bacteria the process of translation initiates when the ribosome assembles on the mRNA at a special ribosome binding site.
The ribosome locates where to bind by a sequence upstream of the first codon (AUG/GUG) that is complementary to a short sequence of the 16S rRNA.
This is known as the Shine-Dalgarno sequence.
This is why bacteria can translate polycistronic mRNA : each coding sequence in polycistronic mRNA is preceded by a Shine-Dalgarno sequence
Note: eukaryotes don’t have this.
Eukaryotic ribosomes recognise 5’ cap (and other bases) at 5’ end of mRNA and thus can only translate the first open reading frame they come to (dictated by the first AUG sequence)
In bacteria the small subunit is accompanied by IF-3 which pr vents the large subunit from attaching until the appropriate time.
Initiator tRNA is brought to the right position by IF-2 along with a GTP (for energy)
IF-2 checks assembly of Met-tRNA and 30 subunit is correct. Initiation process is completed by attachment of the large subunit. IF-1 is also involved at this stage and aids binding of large subunit (50S) and ensures correct assembly of 30 and 50S. Attachment energy attained by IF-2 hydrolysing its bound GTP→GDP. This results in release of initiation factors locking the ribosome in place.
Translation phase 1: initiation (eukaryotes)
In eukaryotes the small subunit, IFs and Met-tRNAi^met find 5’ cap.
Start codon is located by the Kozak sequence (Marilyn Kozak 1943) a consensus sequence code surrounding AUG: ACCAUGG
Lots more IFs are involved.
Poly A tail assists small subunit binding. 5’ cap is bound by several proteins including a complex that helps mRNA being exported from nucleus and then assists with the first round of translation.
Proteins attach to the poly A tail and interact with proteins attached to the 5’ cap. These help improve binding to the small ribosomal unit (forms loop structure).
Consensus sequence - with occasional variations : Kozac solved the problem of how eukaryotic ribosomes detect the position to start translation - ribosomes scan the mRNA searching for the sequence around the start codon
ACC-AUG-G The Kozac sequence is likely to be found near ATG of many genes, variations occur.
This shows consensus for 10,000 human genes
Phase 2: elongation
tRNAs carrying aas need to attach to binding sites in ribosomes.
There are 3 sites in the space between the 2 ribosomal units (left to right) :
E-site: exit site
P-site: peptidyl tRNA site
A-site: Aminoacyl tRNA site
Elongation involves the next AA binding to the A site and then being moved along to the P site where it can be joined to the growing peptide chain.
Each tRNA moves through 3 positions
←E←P←A
The first tRNA is an exception and goes straight to the P site
See diagrams start of notebook 2
Bringing the 2 parts of the ribosome together generated 2 sites at which Aminoacyl tRNA can bind.
The P site is at the stage already occupied by initiator tRNA. Then the next Aminoacyl tRNA binds to the A site. The commonest protein in a bacterial cell is EF-1A (aka EF-TU meaning elongation factor temperature unstable). It complexes with GTP and Aminoacyl tRNAs. There is a pool of these complexes in the cell each trying to load their AA into an empty A site - only the right one can succeed.
Mischarged tRNAs are rejected - codon/anticodon must be correctly matched.
Once current (matching) Aminoacyl tRNA is loaded EF1-A hydrolyses it’s GTP to release energy and allow formation of a peptide bond by the ribozyme peptidyl-transferase - an irreversible process.
EF1B (aka EFTS elongation factor temperature stable) recycles EF1A by removing the GDP allowing free GTP to bind. The EF-1A/GTP can then pick up a fresh Aminoacyl tRNA.
The A site covers the second codon in the open reading frame.
Ribosome moves 5’ to 3’ 3 bases along to move next codon to A site. Aided by EF-2 (EG-G) which again hydrolyses GTP on successful translocation
^All this refers to bacteria but the eukaryotic process is fairly similar
