Lecture 6 Reading Introductory Molecular Biology (BIOM-2131) W2025 PDF

BIOM-2131 Introductory Molecular Biology LECTURE 6 From mRNA to Protein LECTURE OVERVIEW ▪ 6.1 Genetic Code ▪ 6.2 Charged tRNAs ▪ 6.3 Ribosomes ▪ 6.4 Translation, Initiation and Stop...

BIOM-2131 Introductory Molecular Biology LECTURE 6 From mRNA to Protein LECTURE OVERVIEW ▪ 6.1 Genetic Code ▪ 6.2 Charged tRNAs ▪ 6.3 Ribosomes ▪ 6.4 Translation, Initiation and Stop of protein synthesis ▪ 6.5 Antibiotics, Proteosome and PTMs  An mRNA sequence is decoded in sets of three nucleotides 4 different nucleotides in mRNA code for 20 different types of aa in proteins the set of rules by which nt sequence of a gene, through an intermediary mRNA molecule, is translated into the aa sequence of a protein, is known as genetic code mRNA nucleotides are read in triplets triplets provide 64 possible codes triples are called codon each codon – one aa  Genetic code most aa are represented by > than one codon three codons that do not specify any aa act as termination site (stop codon) one codon acts as both initiation codon codon that specifies the aa Methionine Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-29 (partial), p. 256  tRNA molecules match aa to codons on mRNA specific adaptor molecules serve as intermediaries between the codons of mRNA and aa transfer RNA (tRNA) ~ 80nt long 4 short segments form double helical structures each region plays important function anticodon region aa binding region (3’-end) Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-33 (partial), p. 259  tRNA molecules match aa to codons on mRNA the “cloverleaf” undergoes further folding to form a compact, L-shaped structure held together by additional H-bonds between different regions of the molecule Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-33 (partial), p. 259  tRNA molecules match aa to codons on mRNA regions crucial to the function of tRNA in protein synthesis anticodon form by a set of three consecutive nt that bind, through base-pairing, to the complementary codon in an mRNA molecule a short, single-stranded region at the 3’ end of the molecule where the aa that matches the codon is covalently attached to the tRNA Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-33 (partial), p. 259  tRNA molecules match aa to codons on mRNA genetic code is redundant single aa may be specified by several different codons two scenarios: more than one tRNA for many of the aa some tRNA molecules can base-pair with more than one codon some tRNA require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch (wobble) at the 3rd position  Specific enzymes couple tRNA to the correct aa recognition and attachment of the correct aa depend on enzyme aminoacyl-tRNA synthetase must recognize its designated aa and nt in the anticodon loop of tRNA each aa has its own aminoacyl-tRNA synthetase Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-35 (partial), p. 260  Specific enzymes couple tRNA to the correct aa “high-energy” bond between the charged tRNA and aa energy of this bond is later used to link the aa covalently to the growing polypeptide chain Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-35 (partial), p. 260  The mRNA message is decoded on ribosomes ribosome is large complex ribosomal proteins ribosomal RNA (rRNA) bacterial and eukaryotic ribosomes are similar in structure and function composed of one large subunit one small subunit complete ribosome several million Da  Bacterial and eukaryotic ribosomes are similar in structure eukaryotic ribosome ribosome large by weight: molecular 2/3 RNA complexes 1/3 protein Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-35 (partial), p. 260  Ribosomal subunits small ribosomal subunit matches the tRNA to the codon of the mRNA large ribosomal subunit catalyzes the formation of the peptide bonds that covalently link the aa together into a polypeptide chain these two subunits come together on an mRNA molecule near its 5’end to start synthesis of a protein mRNA inches forward in 5’-to-3’ direction ribosome translates nt into an aa sequence  Each ribosome has … binding site for an mRNA molecule three binding sites for tRNA molecules A site - aminoacyl-tRNA P site - peptidyl-tRNA E site - exit Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-38, p. 263  Translation takes place in a four-step cycle a charged tRNA enters C-term of the polypeptide at the A site by base- chain is uncoupled from pairing with the the tRNA at the P site and complementary codon joined to the free aa on the mRNA molecule linked to tRNA in A site Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-39 (partial), p. 263  Translation takes place in a four-step cycle large ribosomal subunit shifts relative to small subunit moves the two bound tRNAs P site → E site Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-39 (partial), p. 263 A site → P site  Translation takes place in a four-step cycle small ribosomal subunit moves exactly three nt along the mRNA ejects the spent tRNA resets the A site for new incoming charged tRNA Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-39 (partial), p. 263 This cycle is repeated each time aa is added mRNA is translated in 5’-to-3’ direction N-terminal end of the protein is made first, aa are added to the C-terminal end of the polypeptide chain Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-39 (partial), p. 263  The Ribosome is a Ribozyme rRNAs form the core of the ribosome and are responsible for ribosome structure and catalytic activity tRNA binding sites formed by rRNAs catalytic site for peptide bond formation peptidyl transferase 23S rRNAs of the large subunit ribosome is a ribozyme ribosomal proteins generally located toward surface help fold and stabilize Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-40, p. 264 the RNA core Ribosomes operate with remarkable efficiency eukaryotic ribosome: adds about 6 amino acids to a polypeptide chain each second bacterial ribosome: adds about 20 amino acids to a polypeptide chain each second  Specific codons in an mRNA signal the ribosome where to start starting translation at the correct site on mRNA sets the reading frame critically important step begins with AUG codon requires a special charged tRNA initiator tRNA always carries methionine (eukaryotes) formyl-methionine (bacteria) Initiation of protein synthesis 1/3 initiator tRNA binds tightly to the P site on the small ribosomal subunit small ribosomal subunit loaded with initiator tRNA binds to the 5’ end of an mRNA molecule the small ribosomal subunit scans the mRNA until it encounters the first AUG Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-41 (partial), p. 265  Initiation of protein synthesis 2/3 when this AUG is recognized by the initiator tRNA several initiation factors dissociate from the small ribosomal subunit binding of large ribosomal subunit and completion of ribosomal assembly Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-41 (partial), p. 265 initiator tRNA is bound to the P site the A site ready for next charged tRNA Initiation of protein synthesis 3/3 Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-41 (partial), p. 265  Protein synthesis in bacteria each bacterial mRNA molecule contains specific ribosome-binding sequence, ~ 6nt long, located few nt upstream of the AUG at which translation is to begin bacterial ribosomes can bind directly to a start codon that lies in the interior of an mRNA as long as ribosome-binding site precedes it by several nt polycistronic Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-42, p. 265  The end of translation is signaled by the presence of one of several stop codons in the mRNA the stop codons are UAA, UAG, and UGA are not recognized by a tRNA do not specify an aa signal the ribosome to stop translation  The end of translation proteins known as release factors bind to any stop codon that reaches the A site on the ribosome this binding alters the activity of peptidyl transferase in the ribosome Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-43 (partial), p. 266  The end of translation water molecule is added to the peptidyl-tRNA instead of aa frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule at this point ribosome releases mRNA dissociates into subunits Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-43 (partial), p. 266  Proteins are produced on polyribosomes synthesis of most protein molecules takes between 20 sec and several minutes multiple ribosomes usually bind to each mRNA molecule being translated polysomes/polyribosomes simultaneous translation of the same mRNA ribosomes may be as close as 80nt apart Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-44 (partial), p. 266 eukaryotes and bacteria  Antibiotics inhibitors of prokaryotic protein synthesis some of these drugs exploit small structural and functional differences between bacterial and eukaryotic ribosomes different antibiotics bind to different regions of the bacterial ribosome  inhibit different steps in protein synthesis Antibiotics that inhibit bacterial protein or RNA synthesis Alberts, et. al, Essential Cell Biology, 6th edition, Table 7-3, p. 267 Controlled protein breakdown helps regulate the amount of each protein proteins vary enormously in their lifespan structural proteins can last for months or even years others for only days, hours or even seconds Controlled protein breakdown helps regulate the amount of each protein proteases enzymes responsible for degrading proteins hydrolyze the peptide bond between aa rapidly degrade proteins whose lifetime must be kept short recognize and remove proteins that are damaged or misfolded Alberts, et. al, Essential Cell Biology, 6th edition, Panel 2-6 (partial), p. 80 Controlled protein breakdown helps regulate the amount of each protein proteosomes large protein machines responsible for breaking down proteins present in cytosol and in nucleus contain central cylinder with protease active sites facing inside of the core and stopers at each end Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-46 (partial), p. 268 Controlled protein breakdown helps regulate the amount of each protein in eukaryotes, proteins marked for destruction are modified by small protein – ubiquitin (polyubiquitin chain) Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-46, p. 268  Many proteins require PTM to become fully active post-translational modification covalent modification more than 100 types most common: phosphorylation and glycosylation Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-47, p. 269 There are many steps between DNA and … … protein folding into correct, 3D shape chaperone proteins steer growing polypeptide along productive folding pathways while preventing proteins from aggregating Alberts, et. al, Essential Cell Biology, 6th edition, Figure 7-48, p. 270  Lecture 6 Reading Chapter 7: From DNA to Protein: How Cells Read the Genome pages 254-276 Upcoming Lecture 7 Reading Chapter 8: Control of Gene Expression pages 277-286  Labs Lab 1: Find info regarding your protein of interest DNA Lab 2: Find info regarding your protein of interest RNA, protein  Labs: Keep in mind Is attendance mandatory? yes Participation during Lab section? 5% of final mark Due time for labs? end of the lab section grace period (due date) _.59 My group is done early and wants to leave? submit your assignment on D2L prior to leaving the classroom  In the future labs Assignment posted ahead of time on D2L GA presentations during - write notes will become available at later time 100% - exceed expectations – do more work as a team to complete assignment together during the assigned lab time aim to complete and submit assignment before the end of the lab period  Lab Assignments Lab 1: not asked to …. asked to do …. Find Info → Image Annotate Image → Figure Figure Legend Figure shows screenshot from NCBI website for xyz. Format? remnants from the past… content = mark Lab Assignments – Common Mistakes 1/2 quality and content of figure legends figure legend format not followed Cell figure number, figure title, etc. screenshots with missing or inadequate annotations not enough not detailed enough figure legend(s) don't refer to annotations in screenshot(s)  Lab Assignments – Common Mistakes 2/2 missing in-text citations for papers informal language typos and grammatical errors answering questions outside of figure legends answers about wrong protein/gene answers not directly relevant to questions: including information about regulation at protein level when question is about DNA promoter region  Lab Assignments- Marks course policy on mark appeals have to wait 24hr before you compose an e-mail to request remark entire assignment will be remarked have one week since your mark is released to request a remark  Lab Assignments Lab 2 act on feedback from your GAs Lab 1 will be released sequentially same amount of time before your next lab tentative release dates for Lab 1: Monday lab (51)  Thursday/Friday Tuesday Labs (52, 53)  Friday/Saturday Wednesday Labs (54, 55, 56)  Saturday/Sunday