Lesson 4 Ribosomes 2020-21 PDF

CELL BIOLOGY Lesson 4 PROTEIN SYNTHESIS AND DEGRADATION : Ribosomes and Proteasomes The Central Dogma of Biology DNA RNA Translation Modern view Classic view Transcription PROTEIN The Central Dogma of Biology states that the coded genetic information contained in DNA is first “transferred” to messen...

CELL BIOLOGY Lesson 4 PROTEIN SYNTHESIS AND DEGRADATION : Ribosomes and Proteasomes The Central Dogma of Biology DNA RNA Translation Modern view Classic view Transcription PROTEIN The Central Dogma of Biology states that the coded genetic information contained in DNA is first “transferred” to messenger RNA (mRNA) through a process called TRANSCRIPTION and is then decoded to produce a protein in a process called TRANSLATION. This “classical” view began to change with the discovery of RNA-dependent DNA polymerases that convert RNA into DNA (present in retroviruses). The Central Dogma of Biology The RIBOSOME is part of the cellular machinery required for the process of TRANSCRIPTION Translation or Protein Synthesis. Proteins are the active players that carry out most cellular functions and their activity, location and abundance is highly Ribosome regulated. Therefore, transcription and translation are tightly controlled TRANSLATION processes. THREE DIFFERENT RNAs ARE REQUIRED FOR PROTEIN TRANSLATION RIBOSOMES ▪ Highly abundant compact particles (the number will vary depending on the cell’s function but a human cell can have up to 10 million ribosomes ). ▪ They were first descrided by George Palade in 1953 who observed them by electron microscopy). They were initially known as “the particles of Palade”. ▪ They are present in the cytosol of all cells except in mature spematozoon and in red blood cells were they are very scarce. ▪ Essential for protein synthesis. ▪ They are always composed of two distinct subunits (large and small) but they have a different size in eukaryotes (80 S) and prokaryotes (70S) Svedbger units (S): sedimentation rate of particles that have been subjected to centrifugation. Depends on size, shape and density Large Subunit Small Subunit Complete Ribosome COMPOSITION OF RIBOSOMES One of the largest and more complex structures in the cell. Ribosomes are formed by RNA (60-65%) and proteins (35-40%) : they are RIBONUCLEOPROTEINS RNA : it is a specific RNA, Ribosomal RNA (rRNA) with catalytic activity (Ribozymes) ▪ Prokaryotic Ribosomes ▪ Eukaryotic Ribosomes 3 rRNAs : 16S (in the small, 30S subunit), 5S and 23S (in the large 50S subunit) 4 rRNAs : 18S in the small subunit (40S), 5S, 5.8S and 28S in the large subunit(60S) The largest rRNAs (28S or 23S) catalyze the formation of peptide bonds : they are ribozymes with peptidyl-transferase activity PROTEINS : mostly basic proteins and with two different functions. ▪ Structural Function: they promote ribosome assembly and stabilize the RNA molecules. ▪ Enzymatic Function: involved in facilitating the proper folding of rRNAs and the adequate positioning of tRNAs. EUKARYOTIC AND PROKARYOTIC RIBOSOMES FUNCTIONAL DOMAINS IN THE RIBOSOME LARGE SUBUNIT A- SITE (Aminoacyl site) : binding of tRNA with amino acid P-SITE (Peptidyl site) : binding of tRNA with peptide E SITE (Exit site) : exit of empty tRNA SMALL SUBUNIT mRNA binding site Ribosome Assembly The mRNA molecule slides between the two ribosomal subunits but the binding site is on the small subunit. FUNCTIONAL DOMAINS IN THE RIBOSOME LARGE SUBUNIT A- SITE (Aminoacyl site) : binding of tRNA with amino acid P-SITE (Peptidyl site) : binding of tRNA with peptide E SITE (Exit site) : exit of empty tRNA SMALL SUBUNIT mRNA binding site Ribosome Assembly The mRNA molecule slides between the two ribosomal subunits but the binding site is on the small subunit. SUBCELLULAR LOCALIZATION Ribosomes are found : CYTOSOL Free in the cytosol Attached to the Rough Endoplasmic Reticulum Attached to the outer nuclear membrane Inside the mitocondria or the chloroplasts. R NUCLEAR MEMBRANE RIBOSOMAL BIOGENESIS Ribosome subunits are assembled in the NUCLEOLUS. Ribosomal proteins and rRNAs are synthesized in different compartments. Ribosomal Proteins are synthesized in the cytosol and then enter the nucleus through nuclear pores. Nucleolar DNA is transcribed to produce rRNA that will be subsequently processed. Ribosomal proteins and RNAs assemble in the nucleolus and form the small and large ribosome subunits. The two subunits exit the nucleus through nuclear pores. Functional ribosomes will be formed in the cytosol TRANSFER RNA (tRNA) AMINO ACID The tRNA molecule has a characteristic cloverleaf structure with three hairpin loops 1) One of these hairpin loops contains a three nucleotide sequence called the ANTICODON, that can recognize and decode a CODON sequence in mRNA. 2) At the 3´end, each tRNA has its corresponding amino acid attached through an ester bond. There is at least one specific tRNA for each one of the 20 amino acids found in proteins. ANTICODON GCC CODON Amino acid Anticodon mRNA TRANSFER RNA (tRNA) tRNAs read the information encoded in mRNA and transfer the appropriate amino acid to the nascent polypeptide chain during protein synthesis. The mRNA sequence is decoded in sets of three nucleotides or CODONS according to the GENETIC CODE. THE GENETIC CODE The genetic code is UNIVERSAL The code is REDUNDANT : most amino acids are specified by more than one triplet There is a unique AUG codon that acts as INITIATION CODON for the synthesis of all proteins. There are three codons that do not code for any amino acid. They are STOP CODONS tRNA AND AMINO ACID ACTIVATION The correct pairing of each tRNA to its amino acid occurs in the cytoplasm and is catalyzed by aminoacyl- tRNA synthetases. Amino Acid + tRNA + ATP ====> aminoacyl-tRNA + AMP + PPi ▪ A high energy ester bond is formed between the 3´OH of the tRNA and the appropriate amino acid. ▪ This reaction requires ATP and involves the formation of an activated amino acid-AMP intermediate. ▪ There are many different aminoacyl-tRNA sinthetases (one for each amino acid and corresponding tRNA) Charged tRNA SYNTHESIS OF PROTEINS : TRANSLATION Translation is always initiated on cytosolic ribosomes regardless of the nature of the translated protein. The process of translation occurs in three stages : 1- Initiation 2- Elongation 3- Termination Activation of amino acids (formation of aminoacyl-tRNA) must occur before translation, and protein maturation and folding will take place after translation. INITIATION ELONGATION TERMINATION The ribosome binds to the mRNA and finds the Start Codon The polypeptide chain is elongated by repeated addition of amino acids The ribosome finds a Stop Codon and the polypeptide is released TRANSLATION FACTORS Proteins that associate with the ribosome, the mRNA or tRNAs and are required to facilitate and regulate the different stages of translation. Role Initiation Proteins that promote the proper association of ribosomes with mRNA Elongation Proteins that help aminoacyl-tRNAs bind to the correct site on the ribosome. Termination Proteins that promote termination of translation and release of the mRNA from ribosomes. Prokaryotes Eukaryotes IF-1, IF-2, IF-3 eIF-1, eIF-2, eIF-3, eIF-4A... EF-Tu, EF-Ts, EF-G eEF-1α, eEF1βγ, eEF-2 RF-1, RF-2, RF-3 eRF-1, eRF-3 TRANSLATION INITIATION Translation initiation involves the formation of a RIBOSOME TRANSLATION INITIATION COMPLEX where the anticodon in the initiator tRNA (Met-tRNAiMet) pairs with the start codon in the mRNA molecule. 1. The Small Ribosome Subunit binds to the mRNA molecule and finds the Start Codon. 2. The first tRNA, the Initiator tRNA (tRNAi-Met ) enters the P site and pairs with the start AUG codon 3. The Large Ribosomal Subunit assembles with the small subunit to form the ribosome Translation Initiation Complex. Large ribosomal subunit Initiator tRNA GTP GDP E mRNA 5  Start codon 3  Small ribosomal subunit 5  A 3  TRANSLATION INITIATION COMPLEX INITIATOR tRNA ▪ The initiator tRNA is different from other tRNAs (it has a different sequence). ▪ The initiator tRNA is not the same in eukaryotes (Methionine-tRNAi) and in prokaryotes (Formyl-Methionine-tRNAi) ▪ All newly synthesized polypeptides start with the amino acid Methionine in eukaryotes and Formyl-Methionine in prokaryotes. IDENTIFICATION OF THE START CODON IN PROKARYOTES : Start codons in bacterial mRNAs are preceded by a specific sequence known as the SHINE-DALGARNO SEQUENCE. This sequence is located a few nucleotides upstream of the initial AUG and is recognized by the 3’end of the 16S ribosomal RNA. This binding helps align the mRNA on the ribosome for translation initation. IDENTIFICATION OF THE START CODON IN EUKARYOTES : Ribosomes bind to the 5´CAP (Methyl Guanosine Triphosphate) of mRNAs and scan the mRNA sequence downstream of the cap until they find an AUG that fits with the KOZAK consensus sequence : ACCAUGG. The sequence between the 5’ end and the start codon is not translated and represents the 5’ untranslated region (5’ UTR) of the mRNA. 5’ Cap KOZAK SEQUENCE ACC G ACC 3’ Poly-A AUG G Open Readin Frames (ORF) Frame 3 GC UUG UUU ACG AAU UA Leu Phe Thr Asn TRANSLATION INITIATION IN PROKARYOTES IF1, IF3 and GTP-bound IF2 bind to the small ribosome subunit. IF2 specifically recognizes the initiator tRNA and the 30S ribosome subunit recognizes the Shine-Dalgarno sequence near the 5’ end of mRNA. The initiator tRNA (tRNAifMet ) and the mRNA bind to the small ribosome subunit Correct base pairing between the anticodon on tRNAi and the start codon (AUG) stimulates IF2´s GTP hydrolysis , an irreversible step in translation initiation. GTP hydrolysis stabilizes the tRNAi –AUG interaction, stimulates the release of Initiation Factors and promotes the association of the large 50 S subunit to form a fully functional ribosome. The initiation complex is formed, with the tRNAi sitting on the P site. TRANSLATION INITIATION IN EUKARYOTES The basic mechanism is similar to that of prokaryotes but more complex, with at least 10 Initiation Factors (eIFs) involved. One of the initiation factors helps recognize the 5’ cap in mRNA. The final Initiation Complex is similar except with Met-tRNAi and with a larger ribosome (80S) In eukaryotes the 5’ Cap (Met-G- 3P) interacts with the 3’ poly A tail through the poly-A binding protein (PABP) and some initiation protein complexes. They form a loop that is maintained throughout the translation process so that when the ribosome reaches the termination site it is close to the 5’ end and can initiate another round of translation. Large Subunit 80S Small Subunit 40S TRANSLATION ELONGATION It is similar in prokaryotes and eukariotes and involves several elongation factors. 1. Binding of aminoacyl-tRNA to locus A. 2. Formation of a peptide bond E 3. Translocation of the ribosome P E A TRANSLATION ELONGATION 1. Binding of aminoacyl-tRNA to locus A. The initiator tRNAi is bound at the P site. The first step in elongation is the binding of the next aminoacyl-tRNA to the A site by pairing with the second codon. The aminoacyl-tRNA is escorted to the A site by the Tu elongation factor (EF-Tu) that is complexed with GTP. Correct pairing of the codon in mRNA and the anticodon in the tRNA induces hydrolysis of the EF-Tu-bound GTP and release of the elongation factor. 2. Formation of a peptide bond A peptide bond is formed between the amino acid bound to the initiator tRNA (f-Met or Met) and the amino acid bound to the tRNA on the A site. The Methionine is transferred to the aminoacyl-tRNA on the A site. The newly formed dipeptide stays bound to the tRNA on the A site while the P site is now occupied by an uncharged, amino acid-free, initiator tRNAi. Peptide Bond Formation The energy required for peptide bond formation is provided by the breakage of the high-energy bond between the amino acid and the tRNA (established during the amino acid activation ) The condensation reaction is catalyzed by an rRNA with enzymatic activity ( Peptidyl-Transferase ) in the large ribosomal subunit : 23S or 28S rRNA. Peptidyl-Transferase rRNA (23S or 28S) TRANSLATION ELONGATION 3. Translocation of the ribosome Translocation of the ribosome involves another GTPbound Elongation Factor and requires GTP hydrolysis. GTP hydrolysis allows the ribosome to slide down to the following codon (towards the 3’ end ) and change the position of the peptidyl-tRNA (tRNA-aa-Met) from the A site to the P site. The uncharged, amino acid-free, initiator tRNAi is now sitting on the E site and the A site is empty. The binding of a new codon-matching tRNA to the A site will induce the release of the tRNAi on the E site. TRANSLATION TERMINATION ▪ The three STOP CODONS (UAG, UAA or UGA) are nonsense codons that have no matching tRNAs with complementary anticodons. ▪ When a stop codon reaches the A site on the ribosome, it is recognized by a Release Factor rather than a tRNA. ▪ The Release Factor (RF or eRF-1) stimulates hydrolysis of the bond between the tRNA on the P site and the polypeptide chain. ▪ The completed polypeptide is released from the ribosome. ▪ The tRNA is discharged and the mRNA and the two ribosomal subunits dissociate. ▪ In eukaryotes the loop structure formed by the mRNA (association of the 5’ and 3’ends ) facilitates the reassociation of the ribosome subunits at the 5’ end and the initiation of a new translation cycle. https://www.youtube.com/watch?v=Ikq9AcBcohAht tps://www.youtube.com/watch?v=KZBljAM6B1s https://www.youtube.com/watch?v=5bLEDd-PSTQ https://www.youtube.com/watch?v=TfYf_rPWUdY POLYSOMES OR POLYRIBOSOMES Each mRNA molecule can be simultaneously translated by multiple ribosomes forming a POLYSOME OR POLYRIBOSOME (in prokaryotes and eukaryotes). Once the first ribosome moves away from the initiation site, another ribosome can bind to the same mRNA and start the synthesis of a second polypeptide chain. Each ribosome in the polysome functions independently from the others and synthesizes a separate polypeptide chain. This strategy significantly speeds up the overall rate of protein synthesis and makes the process much more efficient. POLYSOMES OR POLYRIBOSOMES In eukaryotes the presence of circular polysomes facilitates the rapid recycling of ribosomal subunits When a ribosome completes translation and dissociates from the 3′ end, the separated subunits can readily find the nearby 5′ cap (m7G) and initiate another round of synthesis. This rapid recycling of subunits on the same mRNA contributes to the formation of polysomes and greatly increases translation efficiency. RIBOSOMES AND PROTEIN SYNTHESIS AS THERAPEUTIC TARGETS ✓ Many of our most effective ANTIBIOTICS are compounds that act by inhibiting bacterial, but not eukaryotic, protein synthesis. This specificity is critical to avoid toxicity on human cells. ✓ Inhibiting bacterial protein synthesis will inhibit bacterial growth and proliferation. ✓ STREPTOMYCIN, CHLORAMPHENICOL, TETRACYCLINE and ERITHROMYCIN, are specific for prokaryotic ribosomes and are therefore very effective as anti-bacterial drugs. ✓ Other antibiotics such as PUROMYCIN OR CYCLOHEXIMIDE are not used as therapeutic agents because they act on both prokaryotes and eukaryotes or specifically on eukaryotes. INHIBITORS OF PROTEIN SYNTHESIS ARE USED AS ANTIBIOTICS ANTIBIOTIC STREPROMYCIN Prevents the transition from initiation complex to chain elongating ribosomes PROKARYOTS CHLORAMPHENICOL Blocks PEPTIDYL TRANSFERASE PROKARYOTS TETRACYCLINE Blocks binding of aminoacyltRNA to A-site of ribosome PROKARYOTS ERITROMYCIN inhibits TRASLOCATION PROKARYOTS PUROMYCIN Early termination PROKARYOTS and EUKARYOTS CYCLOHEXIMIDE Blocks PEPTIDYL TRANSFERASE EUKARYOTS DIPHTERIA Blocks TRASLOCATION EUKARYOTS RICIN IRREVERSIBLE inhibitor of PEPTIDYL TRANSFERASE EUKARYOTS TOXINS PROTEASOMES A Proteasome is a eukaryotic multisubunit protease complex that degrades proteins that have been marked for destruction by a small protein called UBIQUITIN. After the first Ubiquitin molecule has been covalently attached to the protein that will be degraded, additional Ubiquitins are linked to the first one forming a poly-ubiquitin chain that is recognized by the proteasome. Proteasomes are found in the cytosol and the nucleus. The timed and controlled degradation of proteins is an essential mechanism to regulate the abundance of specific proteins in the cell. The Ubiquitin-Proteasome pathway plays an important role in several biological processes: ▪ Quality control of proteins ▪ Cell cycle regulation ▪ Elimination of misfolded proteins ( particularly relevant in neurodegenerative disorders such as Hungtinton or Alzheimers) STRUCTURE OF THE PROTEASOME The Proteasome complex shows a barrellike structure with a catalytic nucleus (20S) 19S cap (regulator center) cap made of 4 piled rings with protease active sites in the central pore. 20S catalytic nucleus Two additional outer rings act as entry gates for the proteins to be degraded or as exit gates for the resulting small peptides or amino acids. 19S cap (regulator center) cap The Ubiquitin-Proteasome System Self evaluation codon: AUG contains codons: UAA, UAG, UGA Requires TRANSLATION IN EUKARYOTES ATP GTP ENZYMES Tree types where these are linked SUBUNIT Binding site for SUBUNIT SITE SITE SITE Binding of One binding site to tRNA with tRNA with catalyzes Two sites Binds to codon in made of 3 sites tRNA Binds aas to END eIF1, eIF2, eIF3 eIF4, eIF5 contains Stop codon: UAA, UAG, UGA Start codon: AUG RIBOSOMES AMINO ACIDS mRNA ATP ENERGY ENZYMES tRNA GTP FACTORS Tree types LARGE SUBUNIT SMALL SUBUNIT where these are linked catalyzes Binding site for SITE A SITE P SITE E Binding of One binding site to tRNA with aa tRNA with polypeptide Empty tRNA Binds to codon in made of 3 sites Requires TRANSLATION IN EUKARYOTES PEPTIDYL TRANSFERASE tRNA aminoacylsynthetase Binds aas to 3’ END ANTICODON INITIATION Two sites eIF1, eIF2, eIF3 eIF4, eIF5 ELONGATION EF GTP TERMINATION RF