Translation and Transcription - Biology Lecture Slides

BIOL 239 Translation Textbook 9.2, 9.3 1  Lesson-level learning objectives Explain the process of translation Identify 5’ and 3’ ends of mRNA and tRNA during translation Determine which anti-codons can recognize codons, using wobble rules Explain how various DNA m...

BIOL 239 Translation Textbook 9.2, 9.3 1  Lesson-level learning objectives Explain the process of translation Identify 5’ and 3’ ends of mRNA and tRNA during translation Determine which anti-codons can recognize codons, using wobble rules Explain how various DNA mutations affect proteins Compare and contrast processes in prokaryotes and eukaryotes Define key terms 2 3  Translation The process in which the genetic code carried by mRNA directs the synthesis of proteins from amino acids Requires: mRNA, tRNA with attached amino acid, ribosomes 4  Transfer RNAs (tRNAs) amino acid Short, single-stranded RNA molecules 74 to 95 nucleotides long Each tRNA carries one particular amino acid Base pairing between the tRNA anticodon and an mRNA codon determines where an amino anticodon binds to GGG acid becomes incorporated in a growing polypeptide amino acid binds here anticodon 5  tRNA structure Anticodons are also read from tRNAs can carry modified bases 5’ to 3’ produced by chemical alteration of the A,G,C and U nucleotides anticodon 6 Some tRNAs contain modified bases (don’t memorize these) 7  The genetic code There are 61 codons for amino acids (excluding stop codons) But 61 unique tRNA genes not required I (inosine) recognizes A, U, or C 8 Wobble: Some tRNAs recognize more than one codon 9 Wobble: Some tRNAs recognize more than one codon It doesn’t have to be inosine Don’t memorize, but know how to use the wobble rules modified uridines derivative of cytidine 10 How many tRNAs (minimum #) are required to cover all the codons for valine? 11  The ribosome Site of protein synthesis Complex structure composed of protein and RNA Ribosomes have a large and small subunit 12  The ribosome Sizes of subunits differ between prokaryotes and eukaryotes Prokaryotic small subunit carries a 16S rRNA Eeukaryotic has 18S rRNA 13 14 E. coli 15  Translation (E. coli): Initiation phase ~8 bases upstream of start codon The E. coli Shine-Dalgarno sequence is specifically recognized by complimentary sequences in the 16S rRNA of the 30S subunit. Not all prokaryotes have the same ribosome binding site recognition sequence as E. coli, but many have sequences that 16 are very similar in that they are rich in purines (A and G).  Translation (E. coli): Initiation phase Presence of a nearby downstream AUG codon signals initiation of translation by the addition of a tRNA carrying formylmethionine This tRNA carries the same anti-codon as tRNA carrying unmodified methionine, however the rest of the tRNA is unique and is only used for initiation. Some bacteria occasionally 17 use GUG (valine) as a start codon instead of AUG (e.g. Mycobacterium tuberculosis)  Translation (E. coli): Initiation phase The large 50S ribosomal subunit then binds such that the tRNAFMet is placed in the P site of the ribosome. 18 This completes initiation of translation.  Translation (E. coli): Elongation phase Elongation factors (EFs) escort the next tRNA into the A site of the ribosome Peptidyl transferase catalyses the formation of a peptide bond between the carboxyl (C) terminus of formylmethionine and the amino (N) terminus of the second amino acid 19  Translation (E. coli): Elongation phase As the ribosome moves… - fMet moves from P site to E site - new tRNA enters A site, fMet is released 20 - process repeats  Translation (E. coli): Termination phase Nonsense (stop) codon is encountered (for which there is no tRNA). Release factor recognizes the stop codon, moves into the A site. Polypeptide is released from the C-terminal tRNA. 21 The mRNA, tRNA and ribosomal subunits all dissociate from each other. Eukaryotes 22  Translation (eukaryotes): Initiation phase 40S ribosome subunit recognizes and binds the 5’ methylated cap. It then scans along the mRNA until it reaches initiation sequence (in many eukaryotes, this is only AUG, but includes surrounding sequences in mammals) Initiator tRNA carries Met, not fMet. Rest of translation is similar to prokaryotes. 23 Translation: Initiation phase comparison 24 Polyribosome: a complex of several ribosomes translating from the same mRNA 25 Prokaryotes - no cellular organelles - DNA replication, transcription and translation all occur in the cytoplasm (tightly coupled) Eukaryotes - membrane-bound organelles - compartmentalization - DNA replication, transcription in nucleus - translation in cytoplasm (ER) (uncoupled) 26 Prokaryotes As soon as enough of the transcript is made translation can begin mRNA is very short-lived (seconds) transcription DNA 3’ mRNA 5’ protein Eukaryotes mRNA has to travel out of nucleus to cytoplasm before being translated mRNA is longer-lived (minutes to hours) 27 Posttranslational processing modifications that occur to the protein after translation 28 Posttranslational processing modifications that occur to the protein after translation 29 Posttranslational processing modifications that occur to the protein after translation 30 How mutations affect the products of gene expression new alleles 31 How mutations affect the products of gene expression 32 DNA https://youtu.be/T5gEIViVAPw?si=j-nd7IYIE0GaCnVQ Protein synthesis https://youtu.be/yklIwMea4q8?si=2tyLwwiwaFS2rRC5 A bit of both https://youtu.be/d1UPf7lXeO8?si=Klr5W1SEUrMzY8tI 33  Lesson-level learning objectives Explain the process of translation Identify 5’ and 3’ ends of mRNA and tRNA during translation Determine which anti-codons can recognize codons, using wobble rules Explain how various DNA mutations affect proteins Compare and contrast processes in prokaryotes and eukaryotes Define key terms 34  BIOL 239 Transcription Textbook 9.1, 9.3 1  Lesson-level learning objectives Explain the process of transcription Compare and contrast transcription with DNA replication Given a DNA sequence, write out the RNA sequence Identify 5’ and 3’ ends of DNA and RNA during transcription Describe the post-transcriptional modifications of eukaryotic RNA Define key terms 2 RNA transcript is messenger (m)RNA in prokaryotes; it is processed (cut and ligated) to become mRNA in eukaryotes 3 Image © 2013 Nature Education RNA is similar to single- stranded DNA 4  Transcription 5’ 3’ 3’ 5’ 5’ Polymerization of ribonucleotides (A, U, G, C of RNA) guided by complementary base pairing with DNA Nucleotides are added in the 5’-to-3’ direction Uracil is incorporated in place of Thymine in RNA (both pair with Adenine) 5  Transcription RNA polymerase – the enzyme that catalyzes transcription Promoters – DNA sequences near the beginnings of genes that signal RNA polymerase where to begin transcription Terminators – Sequences in the RNA products that tell RNA polymerase where to stop (encoded by DNA) 6  Transcription RNA polymerase – the enzyme that catalyzes transcription Promoters ≠ Start codon Terminators ≠ Stop codon 7  The steps in transcription: E. coli recognizes the promoter DNA coding/sense/+ strand In contrast to DNA polymerase, RNA polymerase doesn’t need a primer. noncoding/nonsense/- strand 8  The steps in transcription: E. coli DNA helix reforms, displacing the RNA transcript During elongation, another RNA polymerase can initiate 9  Electron micrograph of transcription..so, frame-shifts in 1 gene, do not affect other genes 10  The steps in transcription: E. coli Rho protein binds to RNA sequence that No protein involved. is C-rich/G-poor without GC-rich RNA region secondary structure makes hairpin structure 11 How does transcription compare to DNA replication? What is the same? What is different? Why do these similarities and differences exist? DNA replication 12 Prokaryotic genes: what you see is what you get. mRNA and proteins can be directly deduced from the DNA sequence 13 Prokaryotic genes: what you see is what you get. mRNA and proteins can be directly deduced from the DNA sequence template strand / antisense strand 3’ 5’ 5’ 3’ RNA-like strand / sense strand / coding strand 5’ 3’ 14  Structure of a gene that codes for protein (eukaryote) Gene (DNA) (in nucleus) Transcription (still in nucleus) (in cytoplasm) 15 Image adapted from https://courses.lumenlearning.com/suny-osbiology2e/chapter/rna-processing-in-eukaryotes/  The ends of eukaryotic mRNAs 1. Addition of methylated cap at the 5’ end. Capping enzyme adds a guanidine triphosphate in reverse orientation to the 5’ end after polymerization of the transcript’s first few nucleotides. This G is NOT encoded by the gene! Methyl transferases then add methyl groups to the backward G and to one or more of the succeeding nucleotides in the RNA. Critical for efficient translation. 16  In Prokaryotes : 5’ end of transcript has a triphosphate, rather than a methylated cap In Eukaryotes: Gets connected to 5’ nucleotide in primary transcript 17  The ends of eukaryotic mRNAs 2. Addition of 100-200 Adenosines to the 3’ end, known as the poly-A tail. (NOT encoded by the gene) 11-30 nucleotides downstream of AAUAAA. Cleavage also signals termination of transcription. Promotes export from the nucleus, translation, and inhibits degradation 18  Eukaryotic mRNAs 3. RNA splicing – removal of introns Exons – sequences found in both a gene’s DNA and in the mature mRNA. Exons contain coding sequences for the protein product, and 5’ and 3’ UTRs Introns – sequences found in a gene’s DNA but NOT in the mature mRNA. They are removed from the primary transcript. Intervening sequences. 19  RNA splicing Splicing is usually carried out by a complex known as the 20 spliceosome, although some RNA transcripts are self-splicing Not all eukaryotic genes contain introns! Some contain multiple introns 21  Splicing – Dystrophin gene Gene is 2,5000,000 base pairs in length 22  Why are introns present ? Allow for alternative splicing: Produces different mature mRNA molecules from one gene May encode related proteins with different, partially overlapping sequences 23 Image © National Human Genome Research Institute alternative splicing may contribute to the complexity of mammals Caenorhabditis elegans Homo sapiens Each has ~20,000 protein-coding genes 24  Genome sizes human 3,000,000,000 bp Polychaos dubium 670,000,000,000 bp E. coli marbled lungfish 4,600,000 bp 130,000,000,000 bp 25 Images: https://www.arcella.nl/cell-morphology/ https://tropical-fish-keeping.com/  Lesson-level learning objectives Explain the process of transcription Compare and contrast transcription with DNA replication Given a DNA sequence, write out the RNA sequence Identify 5’ and 3’ ends of DNA and RNA during transcription Describe the post-transcriptional modifications of eukaryotic RNA Define key terms 26