The Central Dogma of Molecular Biology
DNA ==> RNA (transcription)
RNA ==> hn/mRNA ==> protein (translation)
functional RNA ==> DNA (reverse transcription, reverse transcriptase)
RNA ==> snRNA ==> processing
RNA ==> tRNA & rRNA ==> translation
Protein ==> transport & processing
RNA ==> 7S scRNA ==> transport & processing
Prokaryotes & Eukaryotes
In prokaryotes and eukaryotes, each gene can be transcribed by more than one RNA polymerase at once.
In prokaryotes, each transcript can be translated by more than one ribosome at once.
In eukaryotes, each transcript may be translated by more than one ribosome at once.
In prokaryotes, each transcript can contain more than one message, however in eukaryotes, each transcript only contains one message.
In prokaryotes, mRNA can be translated while it is still being transcribed, however this cannot happen in eukaryotes
Prokaryotes & Eukaryotes 2
- transcription separated from translation
- do not occur together
- hn/MRNA processing
- no nuclear membrane in prokaryotes, cytoplasm is not separate from the nucleoid
- transcription is not separated from translation
- may occur together
- different sized ribosomal subunits
- involvement of translation factors
- different/more numerous
- transport and processing
- Golgi body, rough ER
Ribosomes read mRNA and produces polypeptide chains.
Polysome: ribosomes + their polypeptide chains (elongating), moving along one mRNA molecule in the cytoplasm of both eukaryotes and prokaryotes.
rER surface: eukaryotes - individual [ribosome/mRNA] units - large and small subunits only come rogether when translation is occurring
Translation of polycistronic message: prokaryotes only
- operons: where there are co-ordinately controlled genes which are transcribed to give one mRNA rather than one gene.
- 3 messages in one (more/less)
- translation termination signals within which leads to the release of the proteins as individual
- translation immediately starts at the next message along.
- A chain of amino acids
- correct amino acid added each time
- linked by peptide bonds
- convert sequence of nucleotides in the mRNA chain to polypeptide chain (primary structure)
Accuracy of Translation
Cells must maintain accuracy of translation in two ways...
- Make sure that the right amino acid is loaded onto the correct tRNA.
- Aminoacyl tRNA synthetases is the enzyme involved
- Checking that the codon:anticodon pairing is correct
- incorrect pairings fall apart faster than the amino acid can be incorporated into the growing polypeptide chain.
- However, there is a degree of 'WOBBLE' allowed in the system
- anticodon is a loop, not perfectly linear
- can get non-standard base pairings
- nt, anticodon:nt codon
- only certain mispairings are allowed
- degeneracy of the code
- decrease the number of tRNA types needed.
Stages of Translation
- Stages of translation:
- Processing and Transport
- Genetic code read in 3 nt blocks = codons
- Therefore there is a link between the codon (nucleotides) and the amino acid, an adapter molecule, known as tRNA.
- It is important that the correct amino acid is loaded onto its appropriate tRNA. This is known as aminoacylation of tRNA or tRNA charging.
- Managed by enzymes known as:
- aminoacyl tRNA synthetases
- recognises both tRNA and amino acid
- proof reads and then will cleave the amino acid from the tRNA if the incorrect one has been added.
- aminoacyl tRNA synthetases
Reaction: tRNA-AA + ATP + amino acid ==> AA-tRNA-AA + AMP + PPi
Nomenclature: the enzyme is named to take account of the amino acid that it is loading onto the appropriate tRNA. e.g. Seryl tRNA synthetase will catalyse the reaction:
Serine + tRNA-Ser + ATP ===> Seryl-tRNA-Ser + AMP + PPi
First amino acid
In Prokaryotes & Eukaryotes
- AUG, Methionine
- Has a specific tRNAf-Met
- Formyl group added to a methionine (formyl-methionine, fmet)
- All other AUG codons, internal, code for Met with a different tRNA-Met molecule.
- tRNAf-Met is the initiator tRNA - joins during initiaiton, not elongation
Initiation of translation in E.coli
- Uses initiation factors
- IF3 binds to the 30S subunit
- IF3:30S complex attaches to the initiation site on mRNA
- IF2:GTP complex delivers fMet-tRNA-Met to the 30S:IF3:mRNA complex
- 50S subunit joins the 30S:IF3:mRNA:fMet-tRNA-Met complex
- GTP ==> GDP + Pi and IF2:GDP complex and IF3 dissociate
- The end of initiation leaves fMet-tRNA-Met over the first AUG and the next codon alongside
- 2 sites within the ribosome:
- P, peptidyl site (contains AUG:fMet-tRNA-Met)
- A, aminoacyl site (empty)
- Elongation begins when the appropriate AA-tRNA-AA enters the A site
- Reaction is catalysed by peptidyl transferase.
- Formation of a peptide bond between the carboxyl groups of the fMet and the amino group of the next amino acid along
- tRNA deacylase breaks the first fMet-tRNA-Met bond
- ribosomal enzymes also involved
- Ribosom 'slips' along the mRNA
- AA-tRNA-AA moves to the P site
- fMet-tRNA-Met lost
- A sit empty
- Termination codon at A site, complete polypeptide at P site.
- Uses release facters
- Bind RF1 or RF2 plus RF3:GTP
- Complete polypeptide released from ribosome and final tRNA
- RF3:GTP to RF3:GDP + Pi, dissociation of release factors
- Ribosome subunits and mRNA dissociate
Translation in Eukaryotes
The same but for...
- 40S and 60S ribosome subunits
- 40S subunit + mRNA + primary AUG
- No RNA recognition sequence
- uses 5' cap
- scans mRNA for AUG
- need not be the first AUG that is used
- The initiation methionine is NOT formylated
- tRNAi-Met recognises the initial AUG
- Internal AUG recognised by tRNA-Met
- Interactiion with elongation factors
- Initiation requires numerous factors (11+)
- Initiation requires ATP to ADP to scan mRNA and building the initiation complex
- Elongation and termination are not so well understood and the numbrs and function of many of the eukaryotic factors is unclear.
Process/Transportation of Polypeptides
Modification of Proteins
- e.g cleavage and glycosylation
Transport of the final protein
- Inserted into a membrane, secreted, targeted to a specific organelle.
- If this is the case, the polypeptide needs to pass through the rER and golgi.
- the signal that tells the cell is held within the protein itself
- signal sequence, a short sequence of amino acids at the start of the polypeptide: the signal hypothesis.
The Signal Hypothesis
- The polypeptide synthesis begins.
- First few amino acids appear: signal sequence
- Signal sequence recognised and bound by SRP
- Ribosome:mRNA:polypeptide dragged to the rER, polypeptide synthesis is inhibited
- SRP docks to SRP receptor on rER to form the ribosome-translocon complex
- SRP:SRP receptor complex dissociates, polypeptide synthesis resumes, polypeptide extruded into lumen of rER (assisted by peptidyl translocase)
- The signal peptide is removed by signal peptidase.
- Growing protein folds, is modified by ER enzymes and is anchored in membrane.
- The ribosome dissociates and mRNA released
NOTE: there is no difference between a ribosome on the rER and one that is part of a polysome. It is the message they are translating that differs.
Modifications to Eukaryotic proteins
- Remove the signal sequence
- Cleavage to give the active form of the protein
- Addition of nucleotides