Translation

Peptide bond formation: proposed role of the 2'OH group of tRNA

Removal of the 2'-OH of the A residue at the 3' end of the tRNA in the P-site reduces the reaction rate one million times. This figure shows a proposed "proton shuttle" mechanism to explain this.

Prokaryotic RNA polymerase and ribosomes at work on the same mRNA

Ribosome: 2-20 aa/sec RNA Pol: 50-100 nt/sec (DNA Pol: 200-1000 nt/sec). RNA polymerase and ribsome work on the same mRNA at the same time b/c speed of transcription and translation are very similar. Can also have more than 1 ribosome translating the same mRNA being transcribed at the same time

Termination in eukaryotes

The class I RF eRF1 acts like prokaryotic RF1 and RF2, but recognizes all three stop-codons. -The class II RF eRF3-GTP delivers eRF1 to the ribosome. If eRF1 recognizes a stop codon, eRF3-GTP binds to the factor recognition center, leading to hydrolysis of GTP. eRF3-GDP is released and eRF1 moves into the peptidyl transferase center. No ribosome recycling factors in eukaryotes. Apparently, eRF1 together with the ATPase Rli1 take part in the preparation of the ribosome for a new translation event.

Eukaryotic initiation (2)

In eukaryotes, the start codon is found by scanning downstream of the 5' end of the mRNA.The start codon is identified by base pairing with the initiator tRNA. eIF1 is released, eIF5 changes conformation, leading eIF2 to hydrolyze the bound GTP. With GDP bound, eIF2 no longer binds the initiator-tRNA and is released together with eIF5. This allows binding of eIF5B-GTP, promoting binding of the 60 S subunit. -Activity in initiation factors of 48S preinitiation complex uses ATP and allow small ribosomal subunit to scan downstream (5' -> 3') on the mRNA until it meets the translation start codon

Global regulation in eukaryotes Cap binding protiens: eIF4E

In translation initiation, eIF4E binds to the cap, and then to eIF4G. Other proteins, 4E-BPs (binding proteins), compete with eIF4G and act as inhibitors of translation initiation. If 4E-BPs are unphosphorylated, they bind tightly to eIF4E, while phosphorylation of BPs inhibit binding. -mTor, that phosphorylates 4E-BPs, is activated by growth factors, hormones and other factors that stimulate cell division. -If elF4G is outcompeted by 4E-BPs when binding to cap then translation is inhibited. mTor can phosphorylate 4E-BPs which inhibits/inactivates 4E-BPs from binding to elF4E and then elF4G can bind to elF4E and translation can occur... -mTOR will phosphorylate 4E-Bps @ certain times when cell is growing and wants to divide.

Steps in translation

Initiation (different in pro- and eukaryotes) Elongation (very similar in pro- and eukaryotes) Termination (very similar in pro- and eukaryotes)

aminoacyl-tRNA synthetase proofreading

Isoleucyl-tRNA synthetase has an editing pocket near its catalytic pocket. B/c AMP-valine (an AA) is small enough to fit into the editing pocket it is moved form the catalytic pocket to the catalytic pocket and is hydrolyzed, cut out, and released again. The correct AA (AMP-Isoleucyl) is fits perfectly into the binding, catalytic pocket and is too large to fit into the editing pocket so it wont be cut out and released. Summary: Isoleucyl-tRNA synthetase has an editing pocket near its catalytic pocket. AMP-valine is hydrolyzed in this pocket while AMP-isoleucine is too large to fit.

Defective mRNAs are degraded in eukaryotes by translation-dependent mechanisms: Nonstop-mediated decay [EUKARYOTES]

Nonstop-mediated decay: no stop codon, ribosome stalled at end -like the tmRNA in PROKARYOTES. -The ribosome reaches the Poly (A) tail and translates it to amino acids (so a stretch of lysines are added to the end of polypeptide) -If this happens then a number of protiens assemble (Dom34, Hbs1*GTP) and bind to A site....other protiens degrade mRNA and protien

Open reading frames

Open reading frames are streches of triplet codons translated continously into a polypeptide. They are in between start and stop codons. Start codon: AUG, in bacteria also GUG and even UUG Stop codons: UAG, UGA and UAA

Peptide bond formation is catalyzed by ribosomal RNA

Peptide bond formation is not done by ribosomal proteins, only catalyzed by rRNA on large ribosomal unit ...b/c all protiens are on the periphery of the large ribosomal unit.

Preinitiation complex

Preinitiation complex = small subunit of ribsome + initator tRNA + mRNA = preintation complex -> then the large subunit of ribsome binds

RF1 resembles

RF1 resembles a tRNA molecule and can fit into A site. RF has a peptide anticodon (amino acid sequence NOT not nucleotides) which base pairs to stop codon on mRNA via H-bonding.

The ribosome has three tRNA-binding sites

-The A (aminoacyl) site is the binding site for the aminoacylated tRNA, holds the tRNA with the AA that will be linked to the growing polypeptide chain, the new tRNAs come in here -the P (peptidyl) site is the binding site for the peptidyl-tRNA, hold the tRNA linked to the polypeptide...(sometimes) -the E (exit) site is the binding site for tRNA released after transfer of the polypeptide chain to the aminoacyl-tRNA The tRNA binding sites are formed on the interface between the large and the small subunit, and can span the distance between the peptidyl transferase center in the large subunit and the decoding center in the small subunit.

The secondary structure of tRNA: the cloverleaf structure From the cloverleaf to the actual 3D structure

2D = cloverleaf structure 3D=Upside down L shape; U-loop makes contact with D loop, structure is stabilized by H-bonding and base stacking -tRNA is single stranded and folds back on itself -tRNA is hydrophobic, excludes water, leads to base-stacking -can be H-bonding between bases or between sugar phosphate backbone

The polypeptide exit tunnel in the large 50S subunit

The polypeptide exit tunnel in the large 50S subunit Where the growing polypeptide chain exits out of.

Termination Types of Release Factors

[Prokaryotes] RF-1: Recognizes the termination stop codons UAA and UAG RF-2: Recognizes the termination stop codons UAA and UGA RF-3: Stimulates disassioation of RF-1 and RF-2 from ribosome after termination RRF: Ribosome recycling factor; responsible for disassiociating the ribosome subunits after translation has termination [Eukaryotes] eRF-1: Recognizes the stop codons eRF-3: Possibly stimulate disassoication of eRF-1 from the ribsome after termination; possibly causes the ribosome subunits to disassociate after termination of translation

Initiator tRNA

always linked to a methanine (Met) amino acid. In prokaryotes it is fMET. In eukaryotes it is just Met

Translation slippage

ie. Lactose operon in mRNA The ribosome doesn't disassmble at first stop codon (lacZ)...but it moves on (slips) to next open reading frame and iniates translation at next gene (lacY)...two new protiens are created from same ribosome

Translation bypassing

ie. T4 gene 60mRNA: reading frame ends with stop codon but the open reading frame continues...so ribosome bypasses nucleotides after stop codons and continues adding amino acids to same growing polypeptide chain

A polyribosome or polysome

is a chain of many ribosomes translating proteins from one mRNA all @ once...probably why there are not many mRNAs produced in cells -Each ribosome contacts about 30 nucleotides of mRNA, but the large size of the ribosome only allows a density of 1 ribosome for every 80 nt of mRNA. Even a small ORF of 1000 bases can bind more than 10 ribosomes. If not for polysomes, only 10 % of the ribosomes in a cell would be active at a given time, due to the low concentrations of mRNA molecules.

Components of translation:

mRNA (template) Amino acids (units to synthesize a polypeptide) Transfer RNAs Aminoacyl-tRNA synthetases Ribosomes (ribosomal RNAs + ribosomal proteins) Translation factors (initiation, elongation, termination)

Defective mRNAs are degraded in eukaryotes by translation-dependent mechanisms: no-go-mediated decay [EUKARYOTES]

no-go-mediated decay: premature stop codon or secondary structure -ribosome is stalled in middle of mRNA -Uses same protiens to recognize and degrade as nonstop medaited decay

Classes of aminoacyl tRNA synthetases

two classes that attach different amino acids: class I: 1 subunit generally class II: 2 or 4 subunits

In the process, EF-Tu-GTP and EF-G-GTP are hydrolysed to EF-Tu-GDP and EF-G-GDP.

For the factors to participate in a new elongation cycle, GDP must be exchanged with GTP. -For EF-G, the affinity for GTP is much higher than for GDP, so the nucleotide can easily be exchanged (GDP and GTP are just exchanges). -EF-Tu needs the help of an exchange factor, EF-Ts to get new GTP.

Frameshifting

Frameshifting: When a ribosome makes a shift when translating the reading frame (i.e. at some point it moves down 2 nucleotides or moves back instead of 3, and different triplet codons are translated to different AAcids) -ie. Programmed frameshifting dnaX of mRNA: Codes for T subunit of DNA pol and..when a frameshift occurs there is an early stop codon...this is a programmed framshift and is required to produce correct protien

Translational control of the abundance of the transcriptional activator Gcn4 in yeast

Gcn4 activates transcription of genes involved in amino acid biosynthesis. -If there is little amino acids in cell, Gcn4 needs to be active to activate transcription of genes that code for protien involved in amino acids biosyntehsis

Regulation of translation Types

Global regulation in eukaryotes: -(all translation is blocked, i.e. low levels of AA in cells) -Phosphorylation of eIF2 -Inactivation of eIF-4E Gene-specific regulation: Multiple Examples

The elongation steps of translation: a summary. Two helper proteins (elongation factors) are involved. The process is highly conserved between prokaryotes and eukaryotes.

Two elongation factors invovled: EF-Tu and EF-G (prokaryotes); EF1 and EF2 (Eukaryotes)

Structure of messenger-RNA IN prokaryotes

[Prokaryotes] -16S rRNA is part of small ribosomal subunit. -16S rRNA binds by complimentary base pairing to ribosome binding site (RBS) [also called Shine Dalgano sequence) -16S rRNA seuqence is always the same but RBS sequence can vary. -Prokaryotes can control how often ribosome will bind to RBS (how many protiens will be produced) by controlling the complementarity of the RBS to 16S rRNA. -Many prokaryotic RNAs contain two or more ORFs and encode more than one protein. We call them polycistronic RNAs. A polycistronic RNA often encode proteins that perform related functions, such as different steps in the biosynthesis of an amino acid or nucleotide. The ORFs will not always have their own RBS (ribosome binding site). Sometimes the ORFs overlap (most often as the sequence (5'-AUGA), making it possible for the ribosome to start a new translation without letting go of the mRNA. Kozak sequence: AUG start codon is embedded in this sequence and when these nucleotides are present from the Kozak sequence ther eis a more higher frequency of iniation of translation. Many eukaryotic RNAs lack these bases, but their presence increase the efficiency of translation

Eukaryotic RNA polymerase and ribosomes [compartmentalization]

In eukaryotes, mRNA synthesis and protein synthesis take place in different cellular compartments. No need to keep up with RNA polymerase, so the ribosome incorporates 2-4 aa/sec [Ribosome translation is slower in eukaryotes). -RNA polymerase and ribosomes DO NOT work on the same mRNA at the same time

In eukaryotes the mRNA is held in a circle by interactions between initiation factors, primarily eIF4G, and polyA-binding protein.

-Not required to have circle intereactions between IFs and Poly(A) tail binding protiens, but it makes sure the 3' end of mRNA is intact and not subjec to degradation

Termination of translation (2) Dissociation of RF1/2

-RF3/eRF3 bind to RF 1/2 and hydrolyze and releases RF 1/2 itself. -When the peptide chain has been released, the class II release factor (RF3, eRF3) helps the dissociation of RF1/2. The class II proteins are GTP binding/hydrolysing proteins, like EF-G, IF2 and EF-Tu

Three mechanisms ensure correct pairing between tRNA and mRNA

(1) Based on H-bonding between 16s rRNA and the condon-anticondon base pairing of tRNA and mRNA Additional pairing between two adjacent As in 16S rRNA in the A-site and the minor groove of correct base pairs formed between anticodon and the first two bases of the codon. (2) Based on conformation of the tRNA in the A site. If conformation is incorrect (b/c wrong tRNA binding) there will not be movement of tRNA, EF-Tu*GTP on the tRNA will not be able to make contact with the factor binding center, thus the GTP canno tbe hydrolyzed to GDP and the EF-Tu*tRNA is release. To release EF-Tu, its GTP must be hydrolysed. Mismatches in the codon-anticodon pairing alter the position of EF-Tu, preventing its interaction with the factor-binding center and reducing its GTPase activity. (3) Accommodation After release of EF-Tu, the tRNA must rotate the aa towards the P-site in a process called accommodation. Incorrectly paired tRNA cannot rotate in A site and will often dissociate or be released in this process.

The structure of tRNA

(1) Stem structure (called acceptor arm)is where the 5' and 3' ends are... at the 3' end there are always the nucleotides ACC (A is the final nucleotide at the 3' end...this ACC is added by a specific enzyme) The amino acid that is carried by the tRNA is always bound to the ribose of this final terminal adenine nucleotide of ACC @ the 3' end. (2) Pseudouridine loop: always contains pseudouridine (3) D loop (has dihydrouridine) (4) anticodon loop: Has anticodon which base pairs with codons on mRNA. Bracketed by purine in the 3' and uracil in the 5' end (5) Variable loop: Varies in nucleotide length btween different tRNA types...important for binding to the protien that attaches the amino acid to 3' end.

Gene-specific regulation [Prokaryotes] Regulation of prokaryotic translation: Inhibition of the binding of the 30S subunit by masking the RBS Regulation of ribosomal protein expression

(1)-Only if there is not rRNA available for binding does the ribosomal protien then accumlate in the cytosol and eventually bind to its own mRNA b/c no other binding sites are available. excess of ribosomal protien and no/little rRNA = binding of ribosomal protien to mRNA = inhibitory translation = feedback mechanism so that there is no more acculation of ribosomal protien in the cell so that it can eventually bind to rRNA and not mRNA.... Similarities between binding sites on rRNA and mRNA for ribosomal protiens...so ribosomal protiens can bind to 16S rRNA or mRNA that translates ribosomal protiens since the binding sites are so similar. (2) mRNA itself (by complimentary base pairing) can block translation iniation...if mRNA sequences base pair to other mRNA sequences that are close to RBS then a loop structure will occur and it will physically prevent binding of small ribosomal subunit.... Translation machinary can resolve these secondary structures is translation is already occuring on another part of the mRNA that has an RBS.

Initiation in Prokaryotes (1) 16S rRNA alignment

-16S rRNA (part of small subunit of ribosome) has a specific sequence that binds (via complimentary base pairing) to ribosome binding site (RBS) on the mRNA (near 3' end). -In prokaryotes, 16S rRNA interacts with the RBS to position AUG in the P site (but not always as perfectly as in this case)...

Initiation in Prokaryotes (3) Three initiation factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA

-3 Initiation factors: IF1: Prevents binding of tRNA to the portion of the small subunit that will become the A site IF2: A GTPase that interacts with the small subunit, IF1 and fMet-tRNAfMet. Prevents other tRNAs from associating with the small subunit. Also acts as an initial docking site for the large subunit, that activates the GTPase activity IF3: Blocks the small subunit from reassociating with the large subunit. It is bound to small subunit when it is not in use to prevent it binding to large subunit. -Normally, charged tRNAs enter the ribosome in the A site, but during initiation, the charged initiator tRNA enters the P site directly. -Initiation factors (IFs) block all binding sites except the P-site so intiator tRNA binds directly to the P-site which is positioned directly after start codon. -IF1+IF2*GTP+IF3+intiator tRNA + 30S small ribosomal subunit + mRNA = 30S Initiation Complex

Coupling an amino acid to a tRNA molecule

-Amino acids are coupled to tRNA before used in protein synthesis -20 common amino acids, for each AA there is a specific enzyme (Aminoacyl tRNA synthetase) which links AA to tRNA -20 Amino Acids = 20 specific aminoacyl tRNA synthetases, one enzyme for each AA -Theoretically: 61 condon (not including stop codons) so there could be 61 tRNAs bringing 20 AA to ribosome -So the enzymes (which can only link one type of AA) can link one AA to more than one (sometimes 6) different tRNAs [these are called iso-accepting tRNAs, tRNAs with same amino acids but different codon]. Linkage of AA to tRNA happens in two steps: (1) Activation of AA : Formation of an aminoacyladenylylate, a mixed acid anhydride that remains closely associated with the enzyme, is formed by using ATP to link an adenine to an amino acid. One phosphate from ATP and the adenine stay attached to the AA and two phosphates (pyrophosphate) are released (from ATP) and the AA is aminoacyladenylylate. (2) Transfer of the aminoacyl residue to tRNA: the aminoacyladenylylated AA is linked to tRNA (the tRNA is also linked to aminoacyl tRNA synthase enzyme) the enzyme binds the tRNA and specific AA. The AA is always specific to the enzyme, not the tRNA (because multiple tRNAs can have the same AA). The activated (acyladenylylate) AA is used to link it to the 3' end of the tRNA (added to the 3'OH of the adenine nucleotide on the 3' of tRNA). When the AA is added AMP is released (Adenine + phosphate) and can be used in cycle again.

Structure of messenger-RNA IN eukaryotes

-Eukaryotic RNAs only contain one ORF. -No 16S rRNA, but there is 18S rRNA -No ribosome binding site is present in the sequence. -Instead, the 5' cap is used to recruit the ribosome. After binding to the cap, the ribosome scans the mRNA until an AUG is encountered. -The preiniation complex of small ribosomal subunit + iniation factors bind to 5' cap. This will move along mRNA until start codon (AUG) is encountered. -The poly-A tail enhances the level of translation of the mRNA by promoting efficient recycling of ribosomes. Kozak sequence: AUG start codon is embedded in this sequence and when these nucleotides are present from the Kozak sequence ther eis a more higher frequency of iniation of translation. Many eukaryotic RNAs lack these bases, but their presence increase the efficiency of translation

The translocation reaction is stimulated by the elongation factor EF-G and requires GTP hydrolysis.

-Hybrid state: tRNA in the A site has a greater affinity for P site when it is linked to the polypeptide chain (b/c the exit channel for growing polypeptide chain is close to the P-site). So when polypeptide is linked to tRNA in A-site the top fo the tRNA still in A site is bent to the P site where as the bottom is still in the A site. This contraint leads to rotation of the small ribosomal subunit. -EF-G binds to GTP (EF-G*GTP) and moves the tRNA (in the hybrid state) from the A site site to P site (and corresponding polypeptide chain from A site to P site) -The hybrid state is resolved by EF-G. THe EF-G binds to bent tRNA in A site and by binding w/ tRNA it make contact with factor binding center. The GTP on the EF-G is hydrolyzed and this hydrolysis leads to conformational change such that small ribosomal subunit rotates back to original position and the tRNA in the A site moves to P site and EF-G*GDP occupies A site and then is ejected. The ribosome/RNA moves 3 nuclueotides (one codon) and the empty tRNA is ejected. After transfer of the peptide chain, the tRNA in the P-site prefers to bind in the E-site of the large subunit, while the now peptide-loaded tRNA in the A-site prefers the E-site. This is accompanied by a rotation of the small subunit. EF-G-GTP binds to this hybrid state and stabilizes it, but the contact between EF-G-GTP and the factor-binding center leads to hydrolysis of GTP. This changes the conformation of EF-G. "Gates" that separate the A-, P- and E-sites are opened, unlocking the ribosome, and EF-G-GDP is bound to the A-site. The A-site tRNA is moved fully to the P-site, pushing the P-site tRNA to the E-site and then out. The mRNA is moved 3 nucleotides due to the base pairing with the tRNA. The small subunit rotates back, EF-G-GDP no longer will bind to the A-site, and a new aa-tRNA can come in. -The ribosome moves 3 nucleotides (one codon) after translation probably b/c of the tRNAs anticodon...

Comparison between prokaryotic and eukaryotic initiation factors

-IF1 and eIF1 both bind to the A site to prevent interactions with tRNA. -The function of IF2 is split between eIF2 and eIF5B. All three are regulated by GTP/GDP. -eIF3 and eIF1 both bind to the A-site, and both are released upon binding of the large ribosomal subunit.

Eukaryotic initiation (1)

-IFs block all sites but the P-site -43S preiniation complex doesn't contain mRNA; so intiator tRNA that is bound th eP-site doesn't base pair with any translation start site codon. -This is why 5' cap (only in Eukaryotic mRNA) is needed! BEcause eIF4E binds to 5' cap (then eIF4G, eIF4A, then eIF4B) After dissociation of the ribosome, four initiation factors, eIF1, eIF1A, eIF3, eIF5 bind to the small subunit, preventing binding of the large subunit and of tRNA to the A site, analogous to IF1 and 3 in prokaryotes. Initiator-tRNA is escorted to the small subunit by eIF2, a three-subunit GTP-binding protein (the ternary complex) and placed in the P site. In eukaryotes, ribosomes are recruited to mRNA by the 5' cap. Before binding to the ribosome, the cap-binding protein eIF4E binds to the cap. Then eIF4G and eIF4A are recruited, followed by eIF4B. eIF4B activates the RNA helicase activity of eIF4A, which removes secondary structures in the mRNA before the mRNA is delivered to the 43S preinitiation complex to form the 48S preinitiation complex. [43S preinitiation complex] = [small ribosomal subunit + initator tRNA*eIF2*GTP + eIF1, eIF41A, eIF3, and eIF5] 48S preinitiation complex =[43S preinitiation complex] + [mRNA + eIF4E, eIF4G, eIF4A, and eIF4B]

Gene-specific regulation [Prokaryotes] Regulation of prokaryotic translation: Inhibition of the binding of the 30S subunit by masking the RBS

-Invovles regulation of translation fo ribosomal proteins -If protien blocks ribosome binding site it will block iniation of translation -An RNA-binding protien binds near the RBS and the 30S small subunit cannot bind to RBS on mRNA and translation iniation does not occur. -Ribosomal protiens not only bind to rRNA to for ribosomes but also bind to own mRNA (ie. RBS and block translation [though it binds to mRNA with lower affinity])

Initiation in Prokaryotes (2) N-formyl methionine

-N-formyl methionine (fMET, from initiator tRNA) is the first amino acid to be incorporated into a polypeptide chain (but only in prokaryotes!) -After synthesis of the polypeptide, the formyl group is removed by a deformylase. Often, the N-terminal methionine is also removed by an aminopeptidase, as well as one or two additional amino acids

Termination of translation (2) Recycling of Ribosome Ribosome recycling factor (RRF) cooperates with EF-G and IF3 to recycle the ribosome after release of the peptide chain.

-The remaining tRNAs in the E and P sites are taken out by RRF, EF-G and IF3. -IF3 comes in last to block the association of the large and small subunits (after tRNAs are removed by RRF and EF-G) -RRF binds in the A-site by mimicking a tRNA. EF-G-GTP is recruited by RRF and removes the tRNAs in a similar way to what happens in elongation (EF-G binds to RRF in A-site this catalyzes a translocation step and the ribosome moves down a codon and ejects the tRNAs. No tRNAs are in the risomes, the RRF is in the Psite.... Then EF-G-GDP and RRF are released together with the mRNA and IF3 binds to small ribosomal subunit.

tmRNAs rescue stalled ribosomes [PROKARYOTES]

-There are some cases where ribosomes dont complete completely translation -Ribosome can be stalled (ie. secondary structure in mRNA, or if through mutation, translation stop codon isnt present any more in mRNA) -In PROKARYOTES there are mixed transfer, messanger RNAs (tmRNAs). -The tRNA part of the tmRNA will bind to a A site in stalled ribosome (BUT ONLY if the ribosome is at the end fo the mRNA, because tmRNA is too big to normally fit into the ribosome A site if not at the end of mRNA) -The tmRNA, with EF-Tu goes into A site and breaks the bond between the mRNA and ribosome -The mRNA part of the tmRNA is now in the ribosome (replaces the orignal stuck mRNA). The mRNA of the tmRNA codes for some amino acids tha tare linked to the orignial polypeptide chain. -SO the polypeptide chain ends with a specific amino acid sequence tag that is recognized by specific proteases...the proteases will bind these amino acid sequences and degreade the protien

Termination of translation (1) The Process - Polypeptide release

-When a stop codon UAG, UGA, UAA) enters the A-site, no tRNA can recognize any of the stop codons. Release factors (RF) will recognize the stop codons, bind to A site, and trigger a release of the peptide chain. -Top of RF (Class I: RF2 or RF2) is close to tRNA in P site (peptidyl tRNA). -The RF releases polypeptide/protein from ribosome: the top of the RF that is close to the polypeptide chain and tRNA always has a GGQ amino acid motif...this GGQ motif is invovled in hydrolyzing the bonds between the polypeptide chain and the tRNA in the Psite. The bond between the protien and the tRNA is hydrolyzed and th eprotien is released from the ribosome. -There are two classes of RFs. Class I factors recognize the stop codon (in E. coli, UAG is recognized by RF1, UGA by RF2, and UAA by both). These factors bind in the A-site with their peptide anticodon (three amino acids) close to the stop codon. -Stop codons are recognized by class I release factors (RFs). In prokaryotes, RF1 recognizes UAG and RF2 UGA, while the third stop codon, UAA, is recognized by both. In eukaryotes, one single RF recognizes all three. Class II RFs (regulated by GTP binding and hydrolysis) stimulate the dissociation of the class I factors after release of the polypeptide chain.

tRNA elements that are recognized by aminoacyl-tRNA synthetases: the second genetic code

-anticodon loop is not the best for determining/discriminating which AA should be added since mulitle codons code for an AA. The acceptor stem and the anticodon loop are the main parts of the tRNA molecule that are recognized by the aa-tRNA synthetase. Changing one nucleotide in the acceptor stem (the discriminator base) may be enough for the tRNA to be used by another synthetase.

Global regulation in eukaryotes Phosphorylation of eIF2

-eIF2-GTP delivers initiator tRNA to the P-site. -Phosphorylation of the α (alpha) subunit of eIF2 inhibits a GTP-exhange factor for eIF2, called eIF2B, leading to reduced level of eIF2-GTP. -The α subunit of eIF2 is phosphorylated by a number of kinases that are activated by conditions like amino acid starvation, viral infection and elevated temperature. mTor, that phosphorylates 4E-BPs, is activated by growth factors, hormones and other factors that stimulate cell division.

Regulation of ferritin translation by iron

Careful regulation of the iron level in the human body is essential. The iron-binding protein ferritin is the major regulator of the level and acts by storing and releasing iron in a controlled manner. It is critical that the ferritin level responds quickly to changes in the level of free iron in the body. -Ferritin protien binds excess iron in the cell -If no/low iron in cell then threre should not be a lot of ferritin in cell....The levels of ferritin in cell is regulated by translation... -Iron regulatory protien (repressor) binds to iron regulatory sequence on mRNA that codes for ferritin and physically blocks iniation of translation b/c small ribosomal unit cannot assemble @ 5' end. -However if there is a lot of iron in cell, the iron binds to the iron regulatory protien repressor and change the conformation of the repressor so that is can no longer bind to mRNA and ferritin mRNA is translated, and ferritin protein is made to bind to excess iron in the cell.

The ribosome is unable to distinguish between correctly and incorrectly charged tRNAs.

Cysteinyl-tRNA charged with cysteine or alanine is read by the ribosome as cysteine

Elongation steps of translation EF-Tu escorts aminoacyl-tRNA to the A site of the ribosome

EF-Tu: GTP-binding protein with GTPase activity. EF-Tu-GTP binds to aminoacyl-tRNA, EF-Tu and EF-Tu-GDP has little affinity. The GTPase activity is stimulated by the same domain in the large subunit that stimulates the GTPase activity of IF-2 (the factor-binding center). Only after entrance of Aa-tRNA in the A site and formation of a correct codon-anticodon complex will the GTPase activity be stimulated -Initator tRNA is in P-site, aminoacyl tRNA enter A-site, a peptide bond forms between amino acid on iniator tRNA and tRNA in A site. -Growing polypeptide chain is tranfered from tRNA in P-site to tRNA in A site, -Then translocation occurs where tRNA in A site is moved to P site (growing polypeptide chain is thus moved to P-site on translocated RNA from A site). -and the empty tRNA from the P site is moved to the R site. The A site is now free for next tRNA to come in -EF-tu gudes tRNA to A site.

The ribosome: Ribosomal RNAs play both a structural and catalytic role

Most ribosomal proteins are on the periphery of the ribosome. The peptidyl transferase and the decoding centers are composed almost entirely of RNA (50S subunit at the top, with purple proteins and grey RNA, 30S at the bottom with dark blue protein and greenish RNA)

Defective mRNAs are degraded in eukaryotes by translation-dependent mechanisms: Nonsense-mediated decay [EUKARYOTES]

Nonsense-mediated decay -Stop codon (by mutation) introduced into mRNA in the coding region (premature ribosome termination) -B/c the ribosome terminated early there are still protiens on the mRNA (ie. from export out of the nucleus, exon junction complex) because the ribosome did not reach the region of mRNA with the protiens. -The nonsense mediated mRNA decay machinary recognizes these protiens and a number of protiens are recruited to degrade the mRNA and the protien created by the prematurely stopping ribosome

Composition of prokaryotic and eukaryotic ribosomes

Ribosomes: consist of coplex of ribosome RNAs and ribosomal protiens (more rRNA by mass/molecular weight) [Eukaryotic Ribosome] -4rRNAs total -Large 60s subunit: 3 ribsomal RNAs (rRNAs) +49 proteins (5.8S rRNA, 5S rRNA, 28S rRNA) -Small 40S subunit: 1 rRNA + ~33 protiens (18S rRNA) [Prokaryotic Ribosome] -3 rRNAs total -Large 50S subunit: 5S rRNA, 23S rRNA + ~ 34protiens -Small 30S subunit: 16S rRNA + 21 proteins -------- 18S rRNA is functionally similar to 16S rRNA -5S rRNA are similar in eukaryotes and prokaryotes -ribosome in Eukaryotes and prokaryotes function similarily, Eukaryotic ribosomes are just larger. -Although there are far more ribosomal proteins than rRNAs in each subunit, more than two-thirds of the mass of the prokaryotic ribosome is RNA. The ribosomal proteins are small (15 kD on average, while the 16 and 23S rRNAs are large (330 daltons per base, almost 1000 kD for 23S rRNA). -Ribosomes not translating exist in cytosol as indivudal small and large subunits

S is an abbreviation of Svedberg, named after the inventor of the ultracentrifuge, Theodor Svedberg

S = rate of sedimentation in ultracentrifuge, increased mass = increased S

Gene-specific regulation [Prokaryotes] Control of translation by mRNA-specific 4E-BPs: Cup specifically inhibits Oskar translation

The Oskar protein is carefully located to the posterior regions of the Drosophila oocyte prior to fertilization. Oskar mRNA is not produced by the oocyte itself, but by attached nurse cells that deposit the mRNA in the anterior part of the oocyte. Then the mRNA is transported to the posterior part. During transport, translation of the mRNA is prevented by the action of a 4E-BP called Cup. Cup is recruited to the mRNA by another protein, Bruno, that binds to several sequences in the 3'-untranslated region. There is too little of Cup to have an effect on all translation, but this localization to a specific mRNA gives efficient inhibition of translation of that particular mRNA. Similar mechanisms regulate the expression of some other proteins as well.

Antibiotics and translation process

The translation is the target of many antibiotics...they bind to some part of the ribosome and block it so that translation cannot continue. -Some target only Eukaryotic cells, some only prokaryotic cells, some both.

The peptidyl transferase reaction

This transfer of the growing peptide chain from the peptidyl-tRNA [tRNA in P-site] to the aminoacyl-tRNA [tRNA in A-site] is catalysed by the peptidyl transferase center in the large ribosomal subunit -rRNA (23S in prok, 28S in Euk) catalyze the peptide bond formation. -ribosomal RNA are more in the center (near the peptidyl transferase center) and ribosomal protiens are found on the periphery...which is why rRNAs are though to catalyze the peptide bond formation between the amino acid from the tRNA in the A site and the growing polypeptide chain from the tRNA in the P site.