protein synthesis Flashcards
what does the exonic portion contain in mRNA
3 and 5 ‘ utr and coding region
what do exons contain?
coding and non coding sequences
in the coding region, bases are grouped into segments called what?
codons , which are complementary to anti-codons.
what is meant by the genetic code is redundant?
we have a greater capacity within the combination of codons that is required as there are 64 codons but only 20 AA
- consequence there is a redundancy in the system to decode the RNA : this protects against the effects of mutations.
- these variations on the genetic code are called silent mutations ( synonymous nucleotide triplets). This occurs because different codons result in the same amino acid.
- the set of three nucleotides always produce the same amino acid. There are a few exceptions such as the stop codon UGA which can also also code for other amino acids.
- generality of the genetic code with a few variations – evidence for evolution
what are 3 key points about protein synthesisi
- Multi stage process carried out by ribosomes.
- Ribosomes are ribonucleoprotein complexes comprising of both RNA and protein
- Protein synthesis is carried out by two populations of ribosomes, one bound to the endoplasmic reticulum, the other free within the cytosol.
where does protein synthesis occur?
in a cleft between the ribosomal subunits. it is a quick porcess and protein turnover is rapid so the cell needs lots of ribosomes.
what is the composition of eukaryotic ribosomes.
- mammalian ribosomes consist of two subunits 40S and 60S ( which are made out of multiple rRNAs and proteins) which together form the 80s ribosome
- they only come together on the mRNA to perform protein synthesis or translation
- 40 and 60 don’t add up to 80 because s stands for spedburg and is not an additive unit.
- 40s subunit have 18s rRNA (1900 nucleotides) and approx. 33 proteins
- 60s subunits have three RNAs, 5S, 5.8S and 28S (120 , 160 and 4700 nucleotides) and approx 49 proteins.
describe the 80s ribosome.
when the two subunits come together around the mRNA, three active sites are formed within the cleft.
Active protein synthesis only can occur when combing the subunits to form these sites: aminoacyl tRNA binding site ( a site) , peptidyl binding site ( p site) or exit ( e site).
why is met-trnai different to other amino acyl tRNAs?
Initiator tRNA binds to the p site, whereas the other amino acyl tRNAs, including met-tRNA bind to the A-site.
describe eukaryotic translation factors
Proteins directly involved in translation are called initiation or elongation factors and have a name that is systematic
•Initiation factors are call eIF + a number
•For eukaryotic initiation factor 1 , 2 etc
•Elongation factors are called eF + number •for elongation factor 1, 2, etc
•Others associated with a ribosomal subunits
•L or S for large subunit or small subunit eg L22
outline the steps in initiation
1) Number of initiation factors that start the process, eIF4 and eIF5. These recognise and bind to the cap structure
2) Other factors, such as poly (A) binding protein bind to the poly (tail).
3) The association of these proteins with the RNA, results In the circularisation of the RNA .
4) EIF3 (eukaryotic initiation factor 3) binds to the 40S ribosomal sub-unit and EIF2 binds to met-tRNA initiator, along with a GTP molecule
5) The 40S subunit and the met-tRNA, along with their respective initiation factors and GTP, form a single 43S ribosomal sub unit.
6) 43S ribosomal subunit binds to the RNA complex to form a 48S pre-initiation complex
5. Met-tRNA associates with the Peptidyl (P) site.
6) As a result, a process called scanning is initiated. Scanning is the process where the ribosome try’s to find the start codon. This involves the consumption of energy via the hydrolysis of ATP forming ADP and pyrophosphate.
7) Scanning begins from the 5’ site, where RNA translocate along the ribosome, one base at a time, using the consumption of ATP for energy. One ATP for each base that translocate.
8) Met-tRNA recognises the start codon and initiation pauses.
9) dissociation of a number of different initiation factors, including EIF2 AND EIF3. Hydrolysis of GTP to form GDP and pyrophosphate via EIF5. This process allows the joining of the 60s subunit to form the 80s ribosome around the rna strand.
outline the steps in elongation
1) tRNA charging by amino acyl tRNA synthase occurs.
2) EF1 I is involved in the binding of the second Aminoacyl tRNA binding to the A site, hydrolysing GTP. Hydrolysis of the GTP provides energy for a peptide bond to be formed which is catalysed by peptidyl transferase., attaching the elongating chain to the newly introduced amino acid.
3) Hydrolysis of a second GTP will allow for the new peptide chain to translocate along the ribosome, with the aid of elongation factor 2 (EF2), translocase, resulting the tRNA occupying the P and the E site, freeing the A site. GDP and Pi is released and the spent tRNA leaves the ribosome through the exit (E) site, ALLOWING RECHARING OF THE TRNA, requiring more energy from ATP.
4) This tRNA must be recharged before it can be reused. This is done by aminoacyl tRNA synthase, with the consumption of an ATP molecule, releasing AMP and pyrophosphate, as well as the recharged tRNA.
5) The next tRNA can now bind to the free A site, with EF1 and GTP, and the cycle can begin again.
outline the steps in termination
- Occurs when the ribosome encounters stop codon , uag.
- Entry of stop codon to the a site, results not in the acquisition of a tRNA, but of release factor 1, ERF1
- Stimulates hydrolysis of terminal Peptidyl-tRNA AND GTP and the polypeptide chain is released.
- GDP and Pi are released, as is the spent tRNA. The two ribosomal subunits disassociate from the Mrna, allowing recycling of the factors and the protein machinery.
what are polysomes?
association of a single RNA with multiple ribosomes . IN ORDER FOR PROTEIN SYNTHESIS TO EFFICENTLY UTILISE AN MRNA , ONE MESSAGE MAY BE USED BY MANY ACTIVELY TRANSLATIING RIBOSOMES.
what are the energy requirements for charging tRNA ?
1 ATP//AA
what is the energy requirement for polypeptide synthesis
GTP + nATP
What is the energy requirement for the elongation stage
2GTP/AA
what is the energy requirement for termination of polypeptide synthesis
GTP
Describe bacterial ribosomes, what the large and small subunit is and their componants
ribosome - 70s
large and small subunit - 50s +30s
large subunit componants - 23s, 5s RNA, 31 proteins
small subunit componants - 16s RNA, 21 proteins
describe eukaryotic ribosomes, their large and small subunits and their componants
ribosome - 80s large and small subunit - 60 +40s large subunit componants - 28s, 5s, 5.8s RNA + 49 proteins small subunit componants = 18s RNA 33 proteins
what is the cellular target and effect on protein synthesis of streptomycin
small ribosomal subunit, inhibition of initation and misreading of genetic code
tetracyline
small ribsomal subunit, inhibits the binding of the amino acyl tRNA to the ribosome
chloramphenicol
large ribosomal subunit and inhibits the peptidyl transferase activity
erythromycin
large ribosomal subunit, inhibits translocase activity
neomycins
multiple sites, several effects
which factor binds to the 40s ribosomal subunit
EIF3
what charges tRNA ?
amino acyl tRNA synthesase
what is translocation and what does it require?
→tRNAs moving from the A & P sites to the P & E sites respectively.
→uses up one GTP
→ the enzyme translocase and EF2.
the hydrolysis of causes the polypetide to be released in termination?
peptidyl-RNA and gtp
what is produced when tRNA is recharged
→AMP and pyrophosphate
what is involved in the binding of the second amino acyl ?
EF1 and GTP
what do the three active sites stand for ?
aminoacyl tRNA binding site ( a site) , peptidyl binding site ( p site) or exit ( e site).
describe the structure of an mRNA
• At the 5 prime end or start of the RNA there is the Cap structure
• With a 5’ to 5’ linked 7-methylguanosine that has been added, the next two nucleotides also have methylated 2’-postition on ribose ring.
• Together they form the cap structure. This is important for recognition of the mRNA by the translational machinery.
• The central portion is the remnant of the transcript encoded by the gene, specifically the exons. You’ll remember the introns have been removed prior to this.
• The exonic portion includes three different functional parts, the 5’ and 3’ untranslated regions (or UTRs) and the central part, and only part that codes for the protein, the coding region (or CDS)
• The end of the mRNA has the poly A tail. PolyA polymerase adds around 100-250 Adenosines after transcription but before splicing and occurs through a process of Polyadenylation in which recognition of a sequence in the 3 UTR occurs where the RNA is cut and has adenosines added to it.
- In the coding region, the bases are grouped into segments of sequence consisting of codons.
where are anticodons located
within an amino-acylt tRNA that acts as a carrier for a specific amino acid
outline the 3 stages in protein synthesis
- Initiation
- assembly of the ribosome on mRNA together with the first initiator tRNA : aminoacyl tRNA( met-tRNAi). Important : met-tRNAi the initiator is different to other tRNA. Initiator tRNA binds to the p site, whereas the other amino acyl tRNAs, including met-tRNA bind to the A site. - Elongation
- charged tRNAs delivered to the ribosome
- addition of amino acids one at a time building the polypeptide
3.termination
-encounter with a stop codon
end of protein synthesis and release of polypeptide
what is a synonymous/silent mutation ing enes
when a nucleotide changes in the chain but coding remains the same
what is released when a stop codon enters the a site ?
erf1
