Protein Synthesis: Translation

Protein Synthesis - Translation

• Translation is a fundamental and highly conserved process in both prokaryotes and eukaryotes
• It is essential for protein synthesis
• Post-translational modifications and assembly may be required to modify proteins after translation
• Ribosomes facilitate amino acid monomer joining, which is directed by mRNA sequence
• Amino-acyl tRNA synthetases attach amino acids to their corresponding tRNAs
• Amino-acyl tRNA acts as an adapter, converting mRNA's nucleic acid sequence into the protein’s amino acid sequence

Stages of Translation

• Initiation: Translational apparatus components combine with mRNA, and tRNA (with the first amino acid) binds to the start codon
• Elongation: Amino acids are delivered to mRNA via amino-acyl tRNAs and added to the polypeptide chain
• Termination: A stop codon in mRNA is recognized by a protein release factor, disassembling the translational apparatus and releasing the completed polypeptide

Translation Tools

• Ribosome Structure: A (aminoacyl) and P (peptidyl) sites are cavities where charged tRNAs bind during polypeptide synthesis
• E (exit) site: This is where discharged tRNAs exit from the ribosome
• mRNA-binding site: It positions mRNA for the translation of its first codon by binding a sequence near the 5' end of the mRNA
• Location of binding sites: They are near the interface of the large and small ribosomal subunits

tRNA Molecules

• Major loops: Contains three
• Base-paired regions: Contains four
• Anticodon triplet: This is present in all tRNA Molecules
• CCA sequence: Occurs at the 3' terminal and allows for amino acid attachment via an ester bond

tRNA Maturation and Structure

• Nucleotide modifications: These occur during tRNA maturation
• 3D structure: tRNA resembles a hockey stick, with the amino acid attachment site at one end and the anticodon at the other end
• Modified Nucleotides: These are inosine (I), methylinosine (mI), dihydrouridine (D), ribothymidine (T), pseudouridine (¥), and methylguanosine (Gm)

Wobble

• Aminoacyl-tRNA synthetases: 20 types link amino acids to correct tRNAs and some recognize multiple tRNAs due to genetic code redundancy
• Codons vs tRNAs: There are fewer tRNAs than the 61 possible codons
• Codon Variation: Codons encoding same amino acids often differ at the third position
• Wobble Hypothesis: A "wobble" in the base guanine position in the tRNA anticodon allows pairing with uracil instead of cytosine which allows for unusual pairings
• Base Pairing and tRNA numbers: Base pairing at the third codon position enables one tRNA to pair with multiple codons, thus requiring less than 61 tRNAs

Aminoacyl-tRNA Synthetases

• Function: These catalyze the formation of an ester bond between an amino acid's carboxyl group and the appropriate tRNA's 3' hydroxyl (OH) group.
• Charging tRNAs - Step 1: An amino acid and ATP enter the enzyme’s active site
• ATP and Amino acid interaction: ATP loses pyrophosphate, and the resulting AMP binds covalently to the amino acid
• Pyrophosphate processing: It is further hydrolyzed into two phosphate groups
• Charging tRNAs - Step 2: tRNA covalently binds the amino acid, displacing AMP
• Product release: The aminoacyl tRNA is then released from the enzyme

Process of Translation

• Stages: Translation occurs through initiation, elongation, and termination

Messenger RNA (mRNA)

• Function: mRNA carries polypeptide-coding information to the ribosome
• Prokaryotic mRNA: It has a 5' non-coding leader sequence with a ribosome binding site (Shine-Dalgarno sequence) and contains a coding sequence that begins with an AUG start codon, ends with a stop codon (UAA, UAG, or UGA) and a 3' non-coding trailer sequence
• Polycistronic mRNA: This has a gene set for each gene
• Eukaryotic mRNA: It has a 5' cap and a 3' poly(A) tail, but no ribosome binding site (SD site)
• Protein Factors: They are required for translation's initiation, elongation, and termination
• Initiator Amino Acid: N-formylmethionine (fMet) initiates every polypeptide in prokaryotes

Translation Initiation

• Goal: Forming the 70S translation initiation complex
• Step 1 in prokaryotes: Three initiation factors (IF) and GTP bind to the small ribosomal subunit
• Step 2 in prokaryotes: The initiator aminoacyl tRNA and mRNA attach; the mRNA-binding site is part of the 16S rRNA of the small ribosomal subunit
• Shine-Dalgarno Sequence: The 3' end of the 16S rRNA has a pyrimidine-rich stretch that base pairs with this in prokaryotes
• Step 3 in prokaryotes: The large ribosomal subunit joins the complex
• Result: The 70S initiation complex has fMet-tRNAfMet in the P site

Translation Initiation in Eukaryotes

• Difference from Prokaryotes: They use different initiation factors (eIFs), a slightly different assembly pathway, and a non-formylated tRNA met
• eIF2: This binds the initiator tRNA met before the small ribosomal subunit
• mRNA Binding: The complex can attach to the 5' prime cap structure
• Kozak Consensus Sequence: Since there is no Shine-Dalgarno sequence, the ribosome starts translation at an AUG within this sequence
• Poly A tail’s function: It helps set up the initiation complex

Translation Elongation

• Requirements: Elongation needs a peptidyl tRNA or fMet-tRNAfMet (in the first cycle) at the peptidyl (P) site
• Step 1: The second aminoacyl tRNA binds the ribosomal aminoacyl (A) site
• EF-Tu Role: This escorts tRNA to the A site and carries two bound GTPs
• Recycling EF-Tu: GTPs are hydrolyzed when tRNA binds, releasing EF-Tu and EF-Ts helps recycle EF-Tu
• Step 2: A peptide bond forms between the carboxyl group of the terminal amino acid (or fMet in the first cycle) at the P site and the amino group of the new amino acid at the A site
• Peptidyl transferase Activity: The 23S rRNA molecule in the large ribosomal subunit catalyzes the reaction
• Step 3: After EF-G-GTP binds the ribosome and GTP hydrolyzes, the tRNA carrying the elongated polypeptide moves from the A site to the P site.
• tRNA Translocation: The discharged tRNA moves from the P site to the E (exit) site and leaves the ribosome
• mRNA Translocation: The peptidyl tRNA translocates and carries the mRNA with it
• Codon Movement: The next mRNA codon is moved into the A site, which is available for the next aminoacyl tRNA
• Repetition: The entire process repeats for each additional amino acid
• Polysomes: Multiple complexes can form on mRNAs, generating polysomes or polyribosomes

Translation Termination

• Release Factors: Protein synthesis ends through release factors, which recognize the three stop codons and when a stop codon (UAG, UAA, or UGA) is at the A site, it binds
• Polypeptide Release: The protein transfers the polypeptide to a water molecule, releasing it from the tRNA and dissociating the elongation complex components

Heat Shock Proteins

• Molecular Chaperones: They assist proteins for proper folding
• Protein-Folding Diseases: Diseases such as Alzheimer's are sometimes considered this
• Spontaneous Folding: Sometimes the primary sequence of amino acids is enough
• Molecular Chaperone Assistance: Newly made proteins often need molecular chaperones
• Types of Heat Shock Proteins: Hsp70 and Hsp60
• Renaturation: Heat-denatured proteins can be renatured by molecular chaperones during times of stress
• Prion Diseases: These can "self-propagate" due to a misfolded protein causing other versions to misfold

Posttranslational Processing

• Modifications: Many proteins undergo posttranslational processing after the amino acid chain is made
• Processing in Prokaryotes: The N-formyl group is removed from the mature protein, and often methionine and N-terminal amino acids are removed
• Protein hormone insulin: Proinsulin is converted to the active hormone via enzymatic removal of a long internal polypeptide section
• Insulin Chains: The two remaining chains are covalently linked by disulfide bonds connecting cysteine residues
• Protein Splicing: Inteins are removed, and exteins are spliced to form a mature protein

Protein Targeting and Sorting

• Initial Synthesis Location: Begins in the cytosol with nuclear genes
• Ribosome Formation: Large and small ribosomal subunits associate with the 5' end of mRNA which forms a functional ribosome and begins polypeptide synthesis
• Pathway Decision: Once approximately 30 amino acids long, the polypeptide enters one of two pathways

Co-translational Import

• Destination: For endomembrane system compartments
• Process: The forming polypeptide associates with the ER membrane
• Lumen Transfer: Transferred across the membrane into the ER lumen as synthesis continues
• Final Destination: The polypeptide remains in the ER or is transported via vesicles and the Golgi complex

Integral Membrane Proteins

• Insertion: They are inserted into the ER membrane as they are made, rather than the lumen

Polypeptides Destined for Cytosol, Nucleus or Other organelles

• Location of Synthesis: Synthesis continues in the cytosol
• Completion and Release: Upon completion, the polypeptide is released from the ribosome
• Final Location: Polypeptide remains in the cytosol or is transported into the nucleus, mitochondria, chloroplasts, or peroxisomes
• Nuclear Uptake: Occurs via nuclear pores using a mechanism different from that of posttranslational uptake used by other organelles

Mechanism of Co-translational import

• ER Signal Sequence: Proteins destined for the endoplasmic reticulum (ER) initally has an N-terminal peptide
• Signal-Recognition Particle (SRP): The ER signal sequence is bound by it and this complex is a ribonucleoprotein with 6 peptides and a 300 nucleotide RNA molecule
• SRP receptor and GTP Use: The SRP binds to its receptor so the ribosome can dock on the ER membrane, and the nascent polypeptide can enter the pore when the SRP receptor binds GTP
• Translocon: The growing polypeptide translocates through this hydrophilic pore that is created by membrane proteins
• Ribosome Fit: Recent evidence shows that the ribosome fits tightly across the cytoplasmic side of the pore while the ER-lumen side closes off until the polypeptide is ~70 amino acids long
• Signal Peptidase: An enzyme that cleaves signal peptide for protein release into the ER lumen
• Final Steps: The signal peptide remains in the membrane for a time then the ribosome is released and the pore closes

Chaperones and Folding of ER-directed Proteins

• Molecular Chaperones: Folding of newly-made proteins in the endoplasmic reticulum requires them and other folding proteins, such as Bip
• Bip (binding protein): This protein is an Hsp70 chaperone family member and stabilizes hydrophobic protein regions (rich in Trp, Phe, Leu) to allow proper folding instead of aggregation

Protein Disulfide Isomerase

• Function: It catalyzes the formation and breakage of disulfide bonds between cysteine residues in order to make protein folding stable

Transmembrane Proteins

• Integral membrane proteins have two possible insertion methods for those with a single transmembrane segment

Transmembrane Protein - Type 1:

• Insertion: A polypeptide with both a terminal ER signal sequence and an internal stop-transfer sequence is inserted
• Terminal Peptide processing: The terminal peptide is removed, leaving a transmembrane protein
• Orientation: N-terminus remains in the ER lumen and its C-terminus in the cytosol

Transmembrane Protein - Type 2

• Insertion: A polypeptide with a single, internal start transfer sequence is inserted which starts polypeptide transfer and anchors itself permanently in the membrane
• Orientation: The amino-carboxyl orientation of the completed protein depends on the start-transfer sequence's orientation upon entering the translocation apparatus

Multi-Pass Transmembrane Proteins

• Transit sequences: These proteins have multiple internal start and stop transit sequences

Posttranslational Import

• Organelle Entry: This allows some polypeptides to enter organelles after protein synthesis
• Mechanism: Like co-translational import into the ER, import also involves a signal sequence (transit sequence), a membrane receptor, pore-forming membrane proteins, and a peptidase

Mitochondria

• Membrane Spanning: Polypeptides span both the membranes
• Cell-Free experiments: Demonstrated by incubating on ice where polypeptides begin to penetrate but then stall
• Transit Peptidase: This cleaves transit sequence and indicates that the polypeptide's N-terminus is inside
• Proteolytic Enzyme Susceptibility: While that occurs, enzymes added from outside of it can attack what has been inserted
• Final Location: The polypeptide must then span both membranes for a time during import at contact site

Mitochondria/Chloroplasts

• Signal Sequence Recognition: Membrane receptor recognizes the signal sequence directly without cytosolic SRP
• Chaperone Proteins: Furthermore, these play several crucial roles in the mitochondrial process, for example

Chaperone Functions

• Unfolded State: They primarily keep the polypeptide partially unfolded after synthesis in the cytosol, so it binds the transit sequence, and translocation occurs
• Translocation Drivers: These drive and releasing a polypeptide within the matrix, which also depends on ATP
• Final Folding: These help the polypeptide fold into its final conformation

Polypeptide Targeting to Mitochondria

• Polypeptides are synthesized on cytosolic ribosomes but, are destined for the intermembrane space or the inner membrane and it requires two separate targeting sequences (both located at the N-terminus)
• Step 1:The polypeptide is directed to a contact (translocation) site on the mitochondrion by a positively charged or amphipathic transit sequence
• Step 2: A peptidase in the mitochondrial matrix cleaves the transit sequence to uncover a highly hydrophobic second signal sequence
• Step 3: The second signal sequence allows for insertion into the inner membrane, similar to targeting mechanisms for mitochondrial-encoded polypeptides
• Step 4: The remainder of the polypeptide is moved across the membrane into the intermembrane space (or into the inner membrane for integral inner membrane proteins)
• Step 5: A second peptidase cleaves and releases the polypeptide into the intermembrane space

Gene Expression Regulation (Prokaryotes)

Prokaryotic Gene Regulation

• Lac operon regulation: This exhibits dual control with both repression and activation
• Genetic Control Example: Lactose's inducible catabolism in Escherichia coli
• Lactose's Inducible Catabolism. E. coli is a human symbiont
• lac operon components: 3 Structural genes (lacZ, lacY, and lacA), lacI, terminators etc

Lac Operon

• Operon Genes: lacZ, lacY, and lacA are transcribed together
• lacI Gene: It is located near the lac operon and regulates it by producing the lac repressor protein
• Regulatory Elements: Both the regulatory gene and the lac operon itself have promoters where RNA polymerase binds and terminators where transcription stops
• Plac & Operator Site: Place overlaps with where the repressor protein in the active form can bind
• Operon Transcription: The operon's transcription turns into a single long mRNA molecule that codes for all three polypeptides

Lac operon Repression

• Lac Repressor: In absence of lactose, the repressor blocks RNA Polymerase access and prevents transcription in the operator
• Operon State: Resulting transcription is blocked

Presence of Lactose

• Repressor state: It is inactivated, so it cannot bind the operator
• Gene Transcription: Allows transcription of genes to single mRNA

Allolactose

• Form: The isomer of lactose that binds to repressor is allolactose
• Lac repressor state: It is an allosteric protein capable of reversible conversion between two alternative shapes
• Protein Binding State: Protein in form that allow binding occurs when allolactose is absent
• In effector’s presence: The repressor mostly exists in the alternative and inactive state

lac Operon Activation

• Activation: Protein up-regulation through binding of cyclic AMP Receptor Protein to the promoter
• Cyclic AMP (cAMP) Receptor Protein: An allosteric and inactive protien which is activated when binded to cAMP
• Operon Affinity: The cAMP-cAMP complex binds and increases the affinity of RNA polymerase to promote transciption

Active CAP effects

• CAP-cAMP complex: The complex binds to operon recognition site to stimulate transcription
• Promoting RNA binding: It facilitates the promoter being readily bound
• Transcription Process: With the recognition site near the promoter region bounded, RNA polymerase is transcribing of operon

trp Operon Regulation

• The trp operon: An exmaple of genetic control in prokaryotes
• Structure: Includes 5 structural genes (trpE, trpD, trpC, trpB, trpA) plus promoter (Ptrp), operator (O), and leader (L) sequences
• Structural Genes: They're transcribed and regulated as a unit

trp operon

• Repressor protein: This gene is encoded by trpR and inactive when tryptophan is not abundant
• mRNA product: Polycistronic mRNA encodes for the enzymes of the tryptophan biosynthetic pathway
• Repressor Activation: It requires that it be complexed with Tryptophan which will allow repressor to tightly bind

trp leader sequence

• Control Mechanism: Prokaryotes that lack a nuclear message soon after RNA enzyme and nuclear seperate transcription
• Attenuation
• Attenuation is the independent control mechanism via close processing

Attenuation Controlled by trp Leader Sequence

• Nucleotides: trpO transcript encodes 162 nucleotides upstream of start
• Leader mRNA: a section encoding leader peptide of 14 amino acids
• Tryptophan presence affect: when is present in moderate amounts, the sensor peptide is easily made and the long trop operon
• tryptophan Scarcity affect: Peptide is not synthesized, and Synthetic enzymes are synthesized

trp Codons in Regulation

• Position of Tryptophan: two molecules at the sequence are essential in operons
• The leader mRNA: it includes 4 regions capable of base pairing in a hairpin structure
• Riboswitch mechanism

Riboswitch

• Regulatory switch: A section of dependent ability allows regions1, 2, 3, 4 to become secondary structures
• Attenuator: A part of the leader sequence that contains 3 and 4 hairpin structures and it is a string of 8 u molecules
• Hairpin Structure: acts as signal causing transfer of the enzyme
• Coding sequence: A ribosome tranlates when encountering shorteness to codons
• Stalling location: Prevents from forming region

Transcript Enzymes

• Prevents Enzymes and allows other enzymes to produce trytophan

Full availability and base pairing regulation

• With trypthophan avaible , It allows enzymes to translate and pause and prevent binding
• Termination near the the end sequence

Eukaryotic Gene Regulation

Eukaryotic Control

• Gene expression in multiple aspects, which range amplification DNA or RNA to folding protiens

Genomic Control

• Mating-type switching: dependent swapping gene causes and alters strands DNA or RNA which cause splicing

Yeast Mating

• Mating type: Genes swaps to DNA alterence of sequence
• sacchromyces cerevisiae Sexes: Alpha or A
• Chrosome III: Consist three copies of mating info
• SIR: HML aplha or HMRa will copy complete forms due transcribed products

Mating locus

• Type cells: Dependant cells when alpha or beta are removed by copy cassette DNA

Gene codes- Chain reaction

• Genes: For Antibody are rearragements involving segments.
• Tandem array: DINA random array which are removed segments

Chain Sequence

• V and segments from DNA and D and J with segments

Transciption after splicing

• After, DNA splice causes C segment

Eukaryotic Gene Regulation

• mRNA is transcribed by RNA Plymerase II
• Startpoint
• Core promoter helps stimulate and assemble
• control elements vary based on transcription
• The non-coding part which encodes RNA contains control sequence of expression

Primary Transcript

• Addition of end is site which directs the clevage of RNA

Properties of Enchancers

• Recombinants can be used for location and Orientation DNA to study effect

Basal Level

• The Core allows levels
• Without a core promoter, there becomes no transciption
• Enhancer and levels of transciption to alter structure for expression

A model action

• Enhancer model
• Looping DNA for close protien and core promoter

Transcriptional Regulatory Factors.

• Activators
• bending and enchancer are close which makes activator protien bounded in promotor Vicounty
• DNa activators

Activator factors to join complexes

• Factors and RNA to initiate transcription factors.

Transcription Factors (General And Regulatory)

• Gene: genes such as albumin show to controld N A regions like core
• Tissues Cell
• Regulatory factors for brain with non albumine genes

Brain Cell

• Inbrain cell transcript can assemble but lack efficiancy
• Transcription levels from brain

DNA binding

• Motivs Structure to reguate transcription factors
• Alpha and helix
• Loop helix and helix turn to turn and create dimer

Zinc Finger

• A antiparallel and helix with cysteine
• Number zinc contains proteins for zinc

DNA Response squences

• squences which are common which inverted Elements

Hormone Element

• Thyroid contains the same inverse protien squence
• 3 bases
• Absense for Element

Cortsil and Cell Membranes

• Causes
• Binds
• Hormone to release
• Recoeptors to DNA
• Receptor to Dimer
• cAMP level controling gene
• Genes posses factors to protein with factor with binding protien

Eukaroytic Translation

Alternative RNS

• Imunogobiun with forms both
• Single genes that vary heavy

Forms of Production

• Plasma forms
• Exons is added if Exon4 spliced
• Exon4 not spliced but product releases after Exon4

Protein

• Translation