General translation (15) Flashcards
What are the differences in translation in prokaryotes vs eukaryotes?
The fact that prokaryotes have no nucleus has major consequences for mRNA translation. The translation will then be co-transcriptional because there’s no separation between the nucleus and the cytoplasma, meaning that the speed of the translation will have to catch up with the speed of which the mRNA is produced.
In eukaryotes there also has to be extensive processing of the mRNA inside the nucleus (capping, polyadenylation, splicing, additional editing) before export into cytoplasma. This separation leads to translation being significantly slower.
- Co-transcriptional
- Faster translation in prokaryotes
- Processing of mRNA in eukaryotes before export
What is “general” translation, as opposed to specific translation?
General translation is applicable for most RNAs, and is the canonical way to translate
What are the 3 key players that are important for translation/protein synthesis? (names)
- mRNA template
- aa-tRNA (amino acid charged tRNA)
- Ribosome (translation machinery)
mRNA - function
Encodes the order in which amino acids are linked together in the corresponding protein. There is a direct linear relationship between the mRNA sequence and the sequence of the produced protein.
Aminoacyl-tRNA (aa-tRNA) - function
Amino acids attached to the transfer RNAs by aa-tRNA synthetases
Ribosomes - function
Facilitate the correct recognition of codons by aminoacyl-tRNAs and catalyze peptide bonds between amino acids
Mature mRNA template (definition + features)
Processed mRNA present in the cytoplasm.
Translation of mature mRNA starts by initiation of a gene which sets the reading frame.
In addition to the ORF, there are eukaryotic 5’ end 3’ ends with respective untranslated regions, which are important regulatory sequences. 3’UTR tend to be shorter, and most of the regulation goes on the 5’UTR which can be very long.
Circularization of translated mRNA (interactions between which factors? + function)
In eukaryotes, the mRNA is held in a circle by interactions between
- Initiation factors and
- PolyA-binding factors
The circularization increases the translation rate. Most assume that this facilitates ribosomal recycling.
How is tRNA charged by aminoacyl-tRNA synthetases? (general + 2 key steps of charging)
The charging process is catalyzed by amino acid tRNA synthetase ezymes. Each of the 20 tRNA synthetases binds to a specific amino acid, recognizes one or more synonymous tRNAs (encoding the same aa), depending on how many tRNAs are used for encoding that specific aa.
Charging process:
- Coupling of amino acids to AMP forming aminoacyl-AMP. 2 phosphate groups are lost in the reaction.
- Recruitment of uncharged tRNA, where the aa is moved from amicoacyl-AMP to tRNA. The tRNA is now charged as is released from the enzyme, so that the enzyme can take part in another charging cycle.
Prokaryotic ribosome (S-units)
In total: 70S
Two subunits: 50S + 30S
Eukaryotic ribosomes (S-units)
In total: 80S
Two subunits: 60S + 40S
rRNA in ribosomes
In addition to a lot of proteins, ribosomes also contain (noncoding) rRNA which has structural and enzymatic functions.
Ribosomes are composed of approx. 60% rRNA and 40% ribosomal proteins by mass.
rRNA is a ribozyme which makes up about 80% of cellular RNA despite never being translated itself.
Internal structure of the ribosome (tRNA binding sites)
The tRNA binding sites are formed on the interface between the large and the small subunit. The decoding center is located in the small subunit.
A (aminoacyl) site: Binding site for the charged tRNA.
P (peptidyl) site: Binding site for the peptidyl-tRNA (tRNA attached to growing polypeptide chain)
E (exit) site: Binding site for deacylated tRNA (uncharged tRNA is ejected from the ribosome)
Formation of peptide bond
The process is catalyzed by the peptidyl transferase center in the large ribosomal subunit.
The alpha-amino group of the charged aa-tRNA attacks the carbonyl carbon of the peptidil-tRNA (growing polypeptide), leading to peptide bond formation between the two. produce a new one aa longer peptidil-tRNA in the A site, as well as a deacetylated tRNA in the P site. The uncharged tRNA is briefly left in the P site.
Building on A, emptying P.
What are the 3 steps in translation, and are they similar/different in eukaryotes compared to prokaryotes? (short)
Initiation - Different
Elongation - Similar. Same principle, different factors
Termination - Similar
What is special about the initiation step of translation? What is the connection between regulation during initiation and cancer?
The initiation is normally the rate-limiting step of translation. It is the most frequent (not not exclusive) target of regulation.
Cancer is often associated with overproduced initiation factors. The result is a massive production of proteins, including oncogenic proteins driving the transformation into cancer cells.
The initiating methionine (eukaryotes vs. prokaryotes)
In eukaryotes the start codon encodes methionine.
In prokaryotes the start codon encodes N-formyl methionine (fMet). The formyl group is removed during later steps of translation by a deformylase.
In both cases, the N-terminal methionine is often removed by an aminopeptidase when the protein matures, as well as one or two additional aas.
Translation initiation signals in prokaryotes
To dictate which AUG is used and where the ribosome should be recruited to the mRNA sequence:
The Shine Dalgarno sequence (ribosome binding site, RBS) close to the start codon, recruits the ribosome to begin translation. This sequence will pair with the rRNA from the small subunit, recruiting the large subunit.
Translation initiation in eukaryotes
In eukaryotes, the ribosome is loaded from the 5’ end of mRNA. The AUG translation start site AUG is chosen based on the surrounding sequence. There are 7 motifs called the Kozak consensus sequence.
What sequence specify the start codon if the mRNA strand has more than one AUG? Prokaryotes vs. eukaryotes
Prokaryotes: The Shine Dalgarno sequence
Eukaryotes: The Kozak consensus sequence
Cap-dependent initiation: The scanning mechanism (explanation)
The way they imagined it was that the small ribosomal subunit recruited by factors associated with the 5’cap, then the ribosome starts scanning along the 5’UTR sequence until it encountered the initiating AUG sequence (given by Shine Dalgarno or Kozak). The large ribosomal subunit is then recruited, forming a translation competent ribosome which performs elongation and termination.
What are the 5 most important core initiation factors? (names)
eIF2, eIF4E, eIF4A, eIF4G, eIF4F
eIF2 (function)
Forms an eIF2-GTP-Met-tRNA ternary complex (3 subunits) that binds to the 40S subunit, thus mediating ribosomal recruitment of Met-tRNA.
This initiation factor explains why methionine is always the first aa.
eIF4E (function)
Binds to the m7GpppG 5’ terminal “cap” structure of mRNA
eIF4A (function)
RNA helicase, which unwinds complicated structures which can hinder scanning. ATPase and ATP-dependent.
eIF4G (function)
Scaffold connecting various factors. On one hand, it’s associated with the cap binding protein, while on the other PABP. It enhances the helicase activity of eIF4A.
eIF4F (function)
Cap-binding complex, comprising eIF4E, eIF4A and eIF4G. Unwinds the 5’ proximal region of mRNA and mediates the attachment of 43S complexes to it, and assists ribosomal complexes during scanning.
What is the most important auxiliary eukaryotic initiation factor? (name)
PABP = Poly-A binding protein
PABP (function)
Cytoplasmic. Responsible for the circularization of mRNA. Binds to the 3’ poly(A) tail of mRNA and enhances binding of eIF4F to the cap.
The 7 steps during cap-dependent initiation
- eIF2 ternary complex formation, consisting of eIF2, GTP and met-charged tRNA.
- 43S pre-initiation complex formation, through association of the initiation complex with the small ribosome subunit. Cap recognition and circularisation.
- mRNA activation within the Kozak sequence.
- Attachment to mRNA. Ribosomal subunit is recruited to activate mRNA and start scanning 5’ - 3’, until encountering an AUG.
- Initiation codon recognition, hydrolysis of eIF2-bound GTP and Pi, release.
- Ribosomal subunit joining and factor displacement,
- Formation of the elongation competent 80S initiation complex
Initiation in prokaryotes (initiation factor functions + procedure)
The most important prokaryotic initiation factors are IF1, IF2 and IF3.
IF1 is blocking the association of fMet-tRNA with the A site.
IF2 facilitates binding of the met-charged tRNA to the P site.
IF3 prevents association with the large subunit.
One the IFs are bound, the small subunit is ready to bind to tRNA and mRNA. The recruitment is mediated by the SD sequence (ribosome entry site), the start codon is positioned over the P site.
The codon-anticodon base pairing releases IF3 and the large subunit binds. The translation competent ribosome is now formed.
What are the 3 most important prokaryotic initiation factors?
IF1, IF2 and IF3.
IF1 (function)
IF1 is blocking the association of fMet-tRNA with the A site.
IF2 (function)
IF2 facilitates binding of the met-charged tRNA to the P site.
IF3 (function)
IF3 prevents association with the large subunit.
How many different aminoacyl-tRNA synthetases do most cells make?
20, one for each amino acid
How many rRNAs do the large ribosomal subunit contain in prokaryotes and eukaryotes?
2 in prokaryotes, 3 in eukaryotes
Where is the aminoacyl-tRNA incorporated into the ribosome? (start and during elongation)
First time incorporated into P site, then A site for the rest of elongation
What determines the initiating AUG in eukaryotes?
The Kozak consensus
In eukaryotes, does initiation typically occur via the cap-dependent or cap-independent mechanism?
Cap-dependent
Briefly, how is the mRNA circularized?
Via interaction between 5’- and 3’-associated factors
