L-38 Translation (part 2) Nuts and Bolts of Translation + RNA interference

*First Peptide Bond* *The ribosome possesses peptidyl transferase activity.* *-- tRNA positions the second residue of the chain into the catalytic site.* *-- The free amino group on the acyl-tRNA in the ____ site attacks fMet-tRNA in the ____ site.* *-- fMet is transferred to nascent chain in the ____ site forming the first peptide bond.* *-- Uncharged tRNA is left in the ____ site.* *Note: __________ ______________ is the enzyme responsible for the formation of the peptide bond.* *What is the name of the prokaryote subunit responsible for the catalytic activity of peptidyl transferase?* *What is the name of the eukaryotic subunit responsible for the catalytic activity of peptidyl transferase?*

*-- The free amino group on the acyl-tRNA in the ____ site attacks fMet-tRNA in the ____ site.* A site P site *-- fMet is transferred to nascent chain in the ____ site forming the first peptide bond.* A *-- Uncharged tRNA is left in the ____ site.* P you can see how proteins are synthesized from N terminal to C terminal. NiCe. *Note: __________ ______________ is the enzyme responsible for this. * peptidyl transferase *What is the name of the prokaryote subunit responsible for the catalytic activity of peptidyl transferase?* Prok 23S RNA *What is the name of the eukaryotic subunit responsible for the catalytic activity of peptidyl transferase?* Euk: 28S RNA

*miRNA Synthesis* *miRNA genes are transcribed by RNA pol II.* *-- The pri-miRNA stem-loop structure is excised by the _______-__________ complex within the __________* -this complex recognizes and processes the mRNA. *Pre-miRNA is then exported to _________.* *-- Pre-miRNA undergoes a second cleavage by _______(22-25 base pairs).* -works to cut up the mRNA. -Helicase will separate the strands and each strand is loaded into RISC. *-- The single-stranded miRNA or siRNA is then loaded into the ______ ________ __________ _______(RISC). * -nucleophilic protein complex. RISC doesn't work alone, it needs a miRNA to provide target specificity. ------------------------------------------------- *What does miRNA/RISC do? iMperfect binding* *What does siRNA/RISC do? Perfect binding* -- siRNA cleavage requires perfect base pairing between the siRNA and the target mRNA coding sequence.

*-- The pri-miRNA stem-loop structure is excised by the _______-__________ complex within the __________* Drosha-DGCR8 nucleus *Pre-miRNA is then exported to _________.* cytoplasm *-- Pre-miRNA undergoes a second cleavage by _______(22-25 base pairs).* Dicer *-- The single-stranded miRNA or siRNA is then loaded into the ______ ________ __________ _______(RISC). * RNA induced silencing complex (RISC) *What does miRNA/RISC do?* represses translation and increases turnover of target mRNA's *What does siRNA/RISC do?* cleaves a target mRNA with endonuclease activity.

*Eukaryotic Preinitiation Complex* *In eukaryotes, the details of translation initiation varies.* *The major differences are:* *-- __________ brings charged tRNAi to the 40S subunit before mRNA arrives.* *-- _____-__ complex binds to the mRNA 5' ______and brings it to the 40S.* -- The ribosome scans the mRNA 5'→3' to locate the AUG within a Kozak sequence. *-- _____ subunit binds to complete the 80S ribosome.* *In eukaryotes: first thing to bind is ______and not _______. .* Now mRNA comes in, the factor that binds to the 5' cap which just protects the mRNA from exonucleases. It is also necessary for protein translation because eIF-4 binds to the 5' cap. Once eIF-4 has bound, it is loaded to the 40S subunit. *There is no __________ ___________ ____________ in Eukaryotic mRNAs to tell where the start codon is. * *We instead undergo _________where it looks for the start codon. * *Know these, she had alot of questions about the initiation factors and where they bind to.*

*-- __________ brings charged tRNAi to the 40S subunit before mRNA arrives.* eIF-2 (eukaryotic initiation factor 2) *-- _____-__ complex binds to the mRNA 5' ______and brings it to the 40S.* eIF-4 cap *-- _____ subunit binds to complete the 80S ribosome.* 60S *In eukaryotes: first thing to bind is ______and not _______. Bind tRNA at P site first before mRNA gets there.* tRNA mRNA *There is no __________ ___________ ____________ in Eukaryotic mRNAs to tell where the start codon is. * Shine Dalgarno Sequence *We instead undergo _________where it looks for the start codon. *scanning

*Eukaryotic Initiation Complex* Kozak consensus sequence *Translation in eukaryotes initiates at a _______sequence.* *Most often, the first AUG in an mRNA is selected as the translation start site.* -most of the time its the first start codon of the sequence. This is in order to start translation correctly. *EXAM TIP:* *Shine Dalgarno DOESN'T contain AUG while Kozak does contain AUG.* *BOTH have to be recognized by ribosomes to initiate assembly of the ribosomal complex* *Which of the following is true regarding RNA interference?* a. siRNAs are only found in plants b. The synthesis of miRNA and siRNA differ once the pri-miRNAs are transported from the cytoplasm to the nucleus c. miRNA/RISC duplexes are used to cleave mature mRNAs in the cytoplasm d. It is the base pairing between the RNAi's and the RNA Induced Silencing Complex (RISC) that differentiates siRNA from miRNA e. None of the above

*Translation in eukaryotes initiates at a _______sequence.* Kozak *Which of the following is true regarding RNA interference?* e. None of the above

*Eukaryotic Translation is in the Cytoplasm* *Eukaryotic translation is very similar to bacterial translation, but important differences exist.* *-- mRNAs are s________________ and RNA is processed in the ___________.* *-- mRNA is transferred to the ____________ where it may be intercepted by __________-_______.* *Which ribosomal subunit carries out eukaryotic translation?* *Prokaryotic and Eukaryotic translation are mostly the same except for?* a. In prokaryotes, the ribosomal subunits associate only after the first AUG codon is found within the Shine Dalgarno sequence b. In eukaryotes, the mRNA is escorted to the small ribosomal subunit that is already charged with the first tRNAi c. In eukaryotes, finding the AUG within the Kozak sequence results in a charged tRNA being escorted to the P-site d. In prokaryotes, translation begins in the nucleus and ends in the cytoplasm e. Prokaryotic and Eukaryotic translation is the same

*-- mRNAs are s________________ and RNA is processed in the ___________.* synthesized nucleus *-- mRNA is transferred to the ____________ where it may be intercepted by __________-_______.* cytoplasm miRNA-RISC *Which ribosomal subunit carries out eukaryotic translation?* 80S *Prokaryotic and Eukaryotic translation are mostly the same except for?* b. In eukaryotes, the mRNA is escorted to the small ribosomal subunit that is already charged with the first tRNAi

*Major Steps in Protein Synthesis* *1. AA's are activated due to charging of ______.* (already talked about last lecture) *2. Initiation of translation involves binding mRNA and a initiator _______-____.* -- The ribosome is then assembled. *3. It then undergoes elongation of the nascent protein and translocation.* -- A ribosome is a peptidyl transferase. *4. Translation __________ completes the protein.* (all done) *5. Post translational modification* -recognize just like with RNA primary transcript is rarely functional. With translation we have a number of events that happen after the protein is made. -Her exam make protein more functional (post translational modification) *Remember, the primary transcript is rarely ever functional.*

*1. Amino acids are activated due to charging of ______.* tRNAs *2. Initiation of translation involves binding mRNA and a initiator _______-____.* tRNA-met *4. Translation __________ completes the protein.* termination

*RNA Interference (RNAi)* *RNAi is a mechanism active in eukaryotes that involves small ncRNA's (noncoding RNA's) that reduce expression of specific genes. They act as trans elements. They are either called _________ (miRNA) and ________ __________ ________(siRNA) act on mature mRNA in the cytoplasm.* -these guys have mechanisms that impact the flow of genetic information within the cell. We can alter protein expression *Which one regulates both whether an mRNA can be translated and its stability?* *Which one regulates mRNA levels by direct endonuclease cleavage?* *Which one is very useful in the laboratory for "knockdown experiments"?* The way our body regulates proteins by itself.

*RNAi is a mechanism active in eukaryotes that involves small ncRNA's (noncoding RNA's) that reduce expression of specific genes. They act as trans elements. They are either called _________ (miRNA) and ________ __________ ________(siRNA) act on mature mRNA in the cytoplasm.* microRNA (miRNA) small interfering RNA (siRNA) *Which one regulates both whether an mRNA can be translated and its stability?* miRNA *Which one regulates mRNA levels by direct endonuclease cleavage?* siRNA *Which one is very useful in the laboratory for "knockdown experiments"?* siRNA

*Ribosome Translocation* *The ___-____-________ "translocase" moves the mRNA through the cleft in the ribosome.* -clipping down of the mRNA. We end up with the next codon and the next codon and next codon. *-- EF-G-GTP binds near the ____ site.* *-- GTP → GDP results in shifting the ribosome "one" codon along the mRNA.* *-- ____ site is again vacant.* *-- ____ site contains dipeptidyl-tRNA.* *-- _____ site has an uncharged tRNA that will soon dissociate.* (ejector seat) *Important: This process is repeated for each amino acid in the nascent protein.* -- EF-Tu brings the next aminoacyl-tRNA into the A site. -- A new peptide bond is formed.* -- EF-G translocase repositions the next codon into the A site. -- Translation elongation occurs at ~20 residues/sec (super fast)

*The ___-____-________ moves the mRNA through the cleft in the ribosome.* EF-G-GTP translocase *-- EF-G-GTP binds near the ____ site.* A *-- ____ site is again vacant.* A *-- ____ site contains dipeptidyl-tRNA.* P *-- _____ site has an uncharged tRNA that will soon dissociate.* E

*Completion of the Translation Initiation Complex Lehninger * IF-2 hydrolyzes the GTP resulting in dissociation of all IFs. IF-2-GTP ------> IF-2 + GDP + Pi *The ______ subunit swings in and binds, forming the intact _____ S ribosome.* *-- The complex is now a COMPLETE translation initiation complex (small subunit + large subunit):* *-- ___ site is vacant* *-- ____ site has formylmethionyl-tRNAfMet fMet-tRNAfMet* *-- ____ site is vacant * *What is super important to remember about the translation initiation complex?*

*The ______ subunit swings in and binds, forming the intact _____ S ribosome.* 50 S 70 S *-- ___ site is vacant* A site *-- ____ site has formylmethionyl-tRNAfMet fMet-tRNAfMet* P site *-- ____ site is vacant * E site *What is super important to remember about the translation initiation complex?* Formation of this complex is the rate limiting step of translation (formation of proteins)

*Eukaryotic Polysomes Eukaryotic* *Translation _________ and ___________ are essentially the same as in prokaryotes.* *Elongation factors:* -- eEF-1α = EF-Tu -- eEF-1βγ = EF-Ts -- eEF-2 = EF-G *Termination factor:* -- eRF recognizes all stop codons. *Translation initiation occurs in succession as each new ribosome initiates at the Kozak sequence.* *-- Presence of many ribosomes on a single mRNA is a called ___________.* *-- Each ribosome is making a copy of the same protein.* *Note: This is done through the interaction of the 3' poly A tail with the initiation factors. * -The poly-A tail PABP1 helps to stabilize the poly A tail and prevent its degradation.

*Translation _________ and ___________ are essentially the same as in prokaryotes.* elongation and termination Interesting: polio virus, caused by a virus, infect cells and take over the machinery of the cell. Polio virus works by encoding a protein that degrades eIF-4 complex. This prevents us from translation because we don't have any of the translation factor. The virus will then initiate translation in its own way. *-- Presence of many ribosomes on a single mRNA is a called ___________.* polysome

*Prokaryotic mRNA Binds to 30S Subunit* He won't ask us questions on the initiation and elongation factors. IF-1 blocks premature tRNA binding at the A site. IF-3 blocks premature binding of the 50S subunit. *He expects us to know how a peptide is formed and whats going on in the A site and P site. * *What 2 components align in order to allow the P site to be perfect with the start codon?* -*As a result, the AUG start codon is positioned at the ____-site.* *__-___-____ binds charged initiator tRNA (fMet-tRNA) and escorts it into the ___-site.* *mRNA start codon 5' (AUG) 3' base pairs with the fMet-tRNA anticodon. 3' (__ __ __) 5' *

*What 2 components align in order to allow the P site to be perfect with the start codon?* 16S rRNA and the Shine-Dalgarno sequence *As a result, the AUG start codon is positioned at the ____-site.* P-site *__-___-____ binds charged initiator tRNA (fMet-tRNA) and escorts it into the ___-site.* IF-2-GTP P *mRNA start codon 5' (AUG) 3' base pairs with the fMet-tRNA anticodon. 3' (__ __ __) 5' * 3' UAC 5'

*Termination at a Stop Codon* *Translation is terminated when the ribosome reaches a STOP codon*. *What are the 3 stop codons?* *At a stop codon, the ribosome pauses and waits for a new charged tRNA.* *A ___________ _________ (RF) binds in the _____ site.* -- RF-1 recognizes UAG or UAA. -- RF-2 recognizes UAA or UGA. *-- Release factors activate peptidyltransferase hydrolyzing the peptidyl-tRNA bond thus terminating translation.* *-- __________ ___________ __________ (RRF) and ___-___ dissociate the complex.* --IF-3 rebinds to 30S subunit. *Note: Peptidyl Transferase:* 1. Forms peptide bonds 2. Is responsible in forming covalent bond between tRNA and fully grown polypeptide chain forcing the two subunits disassociate

*What are the 3 stop codons?* UAA, UAG, UGA *A ___________ _________ (RF) binds in the _____ site.* release factor A *-- __________ ___________ __________ (RRF) and ___-___ dissociate the complex.* ribosome recycling factor EF-G *-- ___-___ rebinds to 30S subunit.* RF-3

*Prokaryotic 70S Ribosome* *Ribosomes have 3 "tRNA binding" sites:* *What goes in the A site?* *What goes in the P site?* *What goes in the E site?* RF-1 = UAG RF-2 = UGA *Lecture:* We talked about how there is no punctuation between the letters of the codons. It is important that we read the words in the correct spot. If we read in the wrong spot the translation will be off frame and the protein will be misprinted. *Which one of these correctly describes termination in prokaryotes?* a. At the stop codon UGA, Release Factor-1 binds to the ribosome to recruit both Ribosome recycling factor and EF-G and effectively dissociate the complex b. At the stop codon UGA, Release Factor-1 binds to the ribosome to activate peptidyltransferase and effectively release the completed peptide. Recycling factor and EF-G and come in to dissociate the complex c. At the stop codon UGA, Release Factor-2 binds to the ribosome to recruit both Ribosome recycling factor and EF-G and effectively dissociate the complex d. At the stop codon UGA, Release Factor-2 binds to the ribosome to activate peptidyltransferase and effectively release the completed peptide. Recycling factor and EF-G and come in to dissociate the complex e. None of the above

*What goes in the A site?* aminoacyl tRNA *What goes in the P site?* peptidyl tRNA *What goes in the E site?* EMPTY *Which one of these correctly describes termination in prokaryotes?* d. At the stop codon UGA, Release Factor-2 binds to the ribosome to activate peptidyltransferase and effectively release the completed peptide. Recycling factor and EF-G and come in to dissociate the complex

*Antibiotics Target Translation* *What is the name of the specific translation inhibitor from this lecture?* -- It is NOT used clinically. *What exactly does it do?* (mimics) Ribosome peptidyl transferase activity adds puromycin to a nascent polypeptide ending its translation She had a question on puromycin, take a drug and want to prevent a proteins synthesis in the body. Adding a puromycin onto the strand, no more functional protein.

*What is the name of the specific translation inhibitor from this lecture?* Puromycin *What exactly does it do?* It chemically mimics a charged tRNA and enters the A site.

*Shine-Dalgarno Sequence* *What is the significance of the Shine-Dalgarno or Ribosome Binding Sequence (RBS)?* -Shine-Dalgarno sequence is directly upstream of translational start site. -It base pairs with the ribosomes rRNA subunits. -- The RBS is an 8-13 nt purine-rich element in mRNA. *What does the start codon (AUG) encode for in prokaryotes? AKA the first amino acid.* *What about in eukaryotes?* *Although ______ is always the first amino acid in a PROKARYOTIC proteins, the second and each subsequent amino acids can be any one of the 20 AA's.*

*What is the significance of the Shine-Dalgarno or ribosome binding sequence?* Its the site on a prokaryotic mRNA where ribosomes bind to initiate translation. *What does the start codon (AUG) encode for?* N-formylmethionine *Although ______ is always the first amino acid in a prokaryotic protein, the second and each subsequent amino acids can be any one of the 20.* fMet *What about in eukaryotes?* Methionine

mRNA is in a circular fashion. This is because of the initiation interactions. This helps with translation rates. 5' cap and the 3' poly A tail interaction. *That's all he has on translation!* *Which best describes a polysome?* a. Several genes are translated into their respective proteins at the same time b. Each ribosome on a polysome has a different start site and works on different sites of a gene in order to quickly complete translation of one protein c. Each ribosome commences translation at the same Kozak sequence to synthesize their own copy of the same protein d. Polysomes allow for translation to take place in the nucleus of eukaryotes e. Within polysomes, termination takes place at a unique stop codon, UGpseudo-uridine

*Which best describes a polysome?* c. Each ribosome commences translation at the same Kozak sequence to synthesize their own copy of the same protein

*Target mRNA Recognition* *__________ action occurs if there is ideal base base pairing between siRNA and a target mRNA. * -- Usually the target site is located in the coding sequence of an mRNA. *__________ action occurs if the pairing is imperfect. Targets the RISC to come over. * -- Usually the target site is in the 3' UTR of an mRNA. *This complementary action this targets the RISC to the mRNA. The RISC then cleaves up the mRNA. This is how we drive protein expression. By utilizing this mechanism we can routinely decrease protein expression in cells 5 fold. * *During the synthesis of miRNA,* a. Drosha-DGCR8 cleaves a pri-mRNA that will then be exported from the nucleus to the cytoplasm b. RNA helicase separates a cleaved pre-miRNA into two strands that are individually loaded onto RNA Induced Silencing Complex (RISC) c. Dicer cleaves pre-miRNA to prepare it for strand separation via helicase activity d. All of these are correct e. One of these is incorrect

*__________ action occurs if there is ideal base base pairing between siRNA and a target mRNA. * siRNA *__________ action occurs if the pairing is imperfect. Targets the RISC to come over. * miRNA *During the synthesis of miRNA,* d. All of these are correct

*Aminoacyl-tRNAs bind to the A Site for Elongation* *_____________ _____________ (EFs) act on the 70S ribosome.* *____-____-_____ binds an __________-______ in the cytoplasm and delivers this charged-tRNA to the ribosomal ____ site.* *-- Base pairing occurs between the aminoacyl-tRNA anticodon and the second codon in the mRNA.* *What is the purpose of EF-Tu-GTP?* *-- ____ site has an aminoacyl-tRNA* *-- ____ site contains fMet-tRNAfMet* *-- ____site remains vacant* *What is the name of the enzyme that recycles EF-Tu-GDP to EF-Tu-GTP?*

*_____________ _____________ (EFs) act on the 70S ribosome.* Elongation factors *____-____-_____ binds an __________-______ in the cytoplasm and delivers this "charged-tRNA" to the ribosomal ____ site.* EF-Tu-GTP aminoacyl-tRNA A site *What is the purpose of EF-Tu-GTP * It hydrolyzes GTP --> GDP + Pi *-- ____ site has an aminoacyl-tRNA* A *-- ____ site contains fMet-tRNAfMet* P *-- ____site remains vacant* E *What is the name of the enzyme that recycles EF-Tu-GDP to EF-Tu-GTP?* EF-Ts.