HSS2305A - 2024 Lecture 9 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 9 – Gene Transcription and Translation Cont. Today’s Outline Announcements Gene Transcription and Translation REMEMBER: From Genes to Proteins Transcription Synthesis of complementary RNA from a DNA...

HSS2305: Molecular Mechanisms of Disease Lecture 9 – Gene Transcription and Translation Cont. Today’s Outline Announcements Gene Transcription and Translation REMEMBER: From Genes to Proteins Transcription Synthesis of complementary RNA from a DNA template in the nucleus Starting Material: DNA Required machinery: RNA polymerase II Transcription factors End product: messenger RNA (mRNA) Translation Synthesis of proteins in the cytoplasm using information encoded by mRNA Function Starting Material: mRNA Required machinery: Ribosomal RNA (rRNA) and ribosomal proteins Transfer RNA (tRNAs) End product: polypeptide From Genes to Proteins (Complex)  Transcription mRNA Primary transcript Called pre-mRNA or heterogeneous nuclear RNA – hnRNA Initial precursor RNA Equivalent in length to the full length of DNA transcribed (transcriptional unit) Always associated with proteins (not naked) Short lived Or heterogeneous nuclear RNA (hnRNA) 5` 3`  Transcription Coordinating events C-terminal domain (CTD) of RNA polymerase Acts as a scaffold for the organization of factors for pre-mRNAs processing Processing includes: capping, polyadenylation, and intron removal. Machinery for mRNA processing travels with the polymerase as part of a giant “mRNA factory” TFIIH P-TEFb  Transcription Processing mRNA 5’Caps: Capping enzymes recruited by phosphorylated CTD (Ser-5 phorylation by TFIIH) 5’ Cap: Prevents digestion of 5` end from exonucleases Aids in transport of mRNA out of nucleus Plays important role in initiation of translation  Transcription Processing mRNA 5’Caps (7’Methylguanosine cap): GMP(guanosine monophosphate) Last 5’ Pi removed Methyl groups added to added in inverted orientation via terminal guanosine cap and the formation of 5`-5` triphosphate ribose of the adjacent bridge nucleotide  Transcription Processing mRNA 5’Caps (7’Methylguanosine cap): GMP(guanosine monophosphate) Last 5’ Pi removed Methyl groups added to added in inverted orientation via terminal guanosine cap and formation of 5`-5` triphosphate the ribose of the adjacent bridge nucleotide  Transcription Processing mRNA 5’Caps (7’Methylguanosine cap): GMP(guanosine monophosphate) Last 5’ Pi removed Methyl groups added to added in inverted orientation via terminal guanosine cap and formation of 5`-5` triphosphate the ribose of the adjacent bridge nucleotide  Transcription Processing mRNA 3’ poly(A) tail: Poly(A) tail enzymes recruited by phosphorylated CTD: Protects mRNA from premature degradation by exonucleases  Transcription Processing mRNA 3’ poly(A) tail: ~200-250 adenosine residues First an Endonuclease cleaves Adenosine residues added to poly (A) tail and primary RNA ~10-30 added to new 3` end by poly(A)-binding proteins nucleotides downstream from Poly A polymerase attach and stabilize the mRNA AUAAA sequence and help with nuclear export Transcription mRNA https://youtu.be/DoSRu15VtdM Transcription mRNA Processing RNA splicing: Example gene Exons: parts of the gene that contribute to mature RNA product i.e. x, y and z in diagram Intron: Intervening (in between), noncoding sequences Not included in mature RNA product (~ 95% of pre-RNA) i.e. I1, I2 and I3 in diagram Transcription mRNA Processing RNA splicing: Example gene Exons: parts of the gene that contribute to mature RNA product i.e. x, y and z in diagram Intron: Intervening (in between), noncoding sequences Not included in mature RNA product (~ 95% of pre-RNA) i.e. I1, I2 and I3 in diagram  Transcription mRNA Processing RNA splicing: Exon-Intron Exon-Intron Boundary ~-30 10-20 YN Boundary Splice sites: Breaks at the 5’ and 3’ ends Exon-intron boundary sequences are highly conserved: 5’ splice site - G/GU 3’ splice site - AG/G polypyrimidine tract 10-20 YN (Y= pyrimidine, Cytosine or Uracil) branch point sequence (YUNAY) ~ 30 bases upstream of 3` ~15-30% of human inherited diseases estimated to be from errors in splicing https://pmc.ncbi.nlm.nih.gov/articles/PMC8280750/ Exonic splicing enhancers (ESE) Found within exons help recruit Small Nuclear Ribonuclear proteins (snRNPs – “snurps”) composed of small nuclear RNAs bound to specific proteins  Transcription mRNA Processing RNA splicing: Small nuclear ribonuclear proteins (snRNPs – “snurps”) Consist of: small nuclear RNAs (snRNAs; 150 bp) Ex. U1, U2, U4, U5, U6 snRNAs 12+ associated proteins Ex. U1, U2, U4, U5, U6 snRNPs Function: Splicing Ligating pre-RNA into final mRNA product Splicesome macromolecular machine associates with and processes an intron Composed of snRNPs associated proteins Transcription mRNA Processing https://youtu.be/FVuAwBGw_pQ Transcription mRNA Processing RNA splicing: U1 snRNP attaches to 5’ splice site The nucleotide sequence of U1 snRNA is complementary to the 5’ splice site of the pre- mRNA (G/GU) G/GU Transcription mRNA Processing RNA splicing: U2AF U2AF (U2 Auxiliary Factor) protein Binds polypyrimidine tract and the 3’ splice site Attracts U2 snRNP U2 snRNP Attaches to the branch point sequence in the intron Causes a specific adenosine residue to bulge out Become branch point of the “lariat” intron loop  Transcription mRNA Processing RNA splicing: U4/U6 and U5 snRNPs bind to pre-mRNA U4 and U6 snRNAs begin paired to one another U4 snRNA is stripped away from the duplex U6 pairs with U2 snRNA U6 displaces U1 and becomes associated with the 5` splice site  Transcription mRNA Processing RNA splicing: Lariat U6 is a ribozyme Catalyzes cleavage of the 5’ splice site – first cleavage Rxn (Rx1) Forms a free 5’ exon (Exon 1 in the image) Held in place by U5 and bound to 3’ exon (Exon 2) a lariat intron–3’ exon intermediate is formed Transcription mRNA Processing RNA splicing: Second cleavage reaction (Rx2) at the 3’ splice site excises lariat intron ligates two neighboring exons snRNPs released from pre-mRNA Reassembled at the sites of other introns Transcription mRNA Processing Alternative Splicing: Same gene can code for more than one polypeptide. Very important in health and disease. It is estimated that 50-75% of the 25,000 human genes undergo some form of alternative splicing, meaning that most human genes can produce multiple mRNA isoforms, leading to a greater diversity of proteins than would be expected from the number of genes alone. #1-4 = splice variants  Transcription rRNA and tRNA DNA → different types of transcripts mRNA rRNA = ribosomal rRNA Catalyzes amino acid covalent linking during translation Provide structural support tRNA = transfer RNA Matches mRNA nucleotide code to amino acids for polypeptide during translation RNA complex secondary and tertiary structures Base-paired regions form double-stranded stems connected to single-stranded loops contribute to enzymatic activity  Transcription rRNA Eukaryotic cell contains millions of ribosomes > 80% of cells RNA is rRNA 4 distinct ribosomal RNAs Large ribosome subunit (60S)→ 5S, 5.8S, 28S Small ribosome subunit (40S)→ 18S S=Svedberg; sedimentation coefficient; is a measure of how fast a particle, like a protein or ribosome, settles when spun in a centrifuge –influenced by size, shape and mass There are 5 rDNA clusters – clusters of rDNA genes encoding for 100’s of repeats of rRNA that are grouped together on 5 different chromosomes in humans Clusters gathered in nucleoli → factories for ribosome production  Transcription Synthesis of rRNA Clusters of rRNA genes are arranged in tandem Nontranscribed spacers (NTS) Separates transcription units in a ribosomal gene cluster rRNA transcribed by: RNA Pol I (28S, 18S, 5.8S – parts of both the 60S and 40S subunits) RNA Pol III (5S – smallest part of the 60S subunit) – note that 5S is not made in the clusters and is created outside of the nucleoli rRNA transcription has a “Christmas tree” pattern  Transcription Processing rRNA Processing takes place in nucleoli (plural): Assembly of rRNAs occurs in the nucleolus along with corresponding r-proteins that are imported into the nucleus. Fibrillar center (fc) contains rDNA The 5S rRNA genes located outside the nucleolus, transported to the nucleolus Dense fibrillar component (dfc) contains the pre-rRNA transcripts Granular component (gc) contains ribosomes at various levels of assembly Pre-rRNA contains large numbers of methylated nucleotides and pseudouridine residues which play crucial roles in the structural stability and function of ribosomes Assembled 60S or 40S ribosomes Exported through the nuclear pore complexes to the cytoplasm remain free or form rough endoplasmic reticulum (RER)  Transcription Processing rRNA 120` Small nucleolar ribonucleoproteins (snoRNPs) (not the same as Small nuclear ribonuclear proteins (snRNPs) involved in splicing mRNA) Particles that consist of small nucleolar RNAs (snoRNAs) and associated proteins Play active role in processing pre- rRNA in nucleolus to help the structural stability and function of ribosomes > 200 box H/ACA snoRNAs → determine which uridines will be convertd to pseudouridines Uridine is the nucleoside that is composed of the Uricil base and ribose sugar box C/D snoRNAs → determine which nucleotides in the pre-rRNA will have a ribose methylated Transcription tRNA Capable of binding: Its anticodon can bind a specific amino acid i.e. a particular codon (3 nucleotide sequence) in the mRNA Between 73-93 nucleotides in length ~ 50 different species of tRNA ~ 500 tRNA genes in humans may vary in sequence but have same anticodon Where are they transcribed? tRNA genes in small clusters scattered around the genome arranged in tandem Nontranscribed spacer (NTS) Separates transcription units in a tDNA gene cluster tRNAs have promoter sequences within the coding region of the gene Transcribed by RNA pol III (like the 5S rRNA) tRNA precursor is trimmed and numerous bases are modified REMEMBER: From Genes to Proteins Transcription Synthesis of complementary RNA from a DNA template in the nucleus Starting Material: DNA Required machinery: RNA polymerase II Transcription factors End product: messenger RNA (mRNA) Translation Synthesis of proteins in the cytoplasm using information encoded by mRNA Function Starting Material: mRNA Required machinery: Ribosomal RNA (rRNA) and ribosomal proteins Transfer RNA (tRNAs) End product: polypeptide  Translation Genetic Code mRNA Codons Codon nucleotide triplets Matched with amino acid On mRNA Anti-Codons Sequence on tRNA that are complementary to Codon  Translation Codon Degeneracy 20 Amino Acids but 61 sense codons (excludes stop codons) Multiple codons for any amino acid Punctuation: * Called “Redundancy” or “Degeneracy” in genetic code Minimizes chance of wrong amino acid if mutations occur Called “synonymous mutations” Degenerate code: tRNA isoacceptors: UUA tRNAs with unique anticodon UUG that bind same amino acid CUU (see chart) However, cells have ~50 tRNA species despite there being 61 codons Wobble Pairing!  Translation Wobble Pairing First two positions of codon- anticodon pairing is essential Exact base pairing of the third position less critical – “Wobble” Typically pair with either: purine (G or A) pyrimidine (U or C) as appropriate Example, the double-ringed G can pair with either a single- ringed U or C. Some wobble positions can pair with any of the four bases.  Translation Genetic Code - Mutations Codon Degeneracy and Wobble Pairing Reduces effects of DNA mutations on final AA (amino acid) sequence Ser-Iso-Cys Ser-Iso-Cys Ser-Met-Cys Ser-Iso-Stop* Ser-His-Leu Ser-Ala-Ser Ser-Ser Ser-Leu Translation http://youtu.be/5bLEDd-PSTQ  Translation tRNA Formation Amino Acid (AA) Activation: Aminoacyl-tRNA synthetases (AARS) Covalently link AA to the 3` end of their respective tRNA amino acyl-AMP Exactly one for each of the 20 amino acids not like tRNAs ATP required activate the amino acid transferred to the tRNA molecule ATP + amino acid →amino acyl-AMP + PPi amino acyl-AMP + tRNA → aminoacyl-tRNA + AMP  Translation Initiation Process Initiation: Different process in prokaryotic and eukaryotic cells Step 1: ~ 12 eukaryotic initiation factors (eIFs) bind to Small ribosomal subunit (i.e. 40S subunit) Step 2: initiator tRNA-Met enters P site (Peptidyl site) of 40S rRNA subunit in association with eIF2-GTP P A 1)  Translation Initiation Process Initiation: Different process in prokaryotic and eukaryotic cells Step 1: ~ 12 eukaryotic initiation factors (eIFs) bind to Small ribosomal subunit (i.e. 40S subunit) Step 2: initiator tRNA-Met enters P site (Peptidyl site) of 40S rRNA subunit in association with eIF2-GTP P A 2)  Translation Initiation Process Initiation: Step 3: Now the 43S complex (its getting bigger), it will bind to the 5` cap (i.e. the 7-methylguanosine (m⁷G)) of mRNA Aided by several eIFs bound to the mRNA eIF4E → bound to 5` cap eIF4A → removes mRNA double stranded regions eIF4G → links 5` cap to 3` poly(A) tail (circular message) 3)  Translation Initiation Process Initiation: Step 4: The 43S complex scans mRNA in search of initiation codon (AUG 4) – Encodes Met) In eukaryotes ribosomes scan for Kozak sequence (5` ACCAUGG 3`) Step 5: GTP bound to eIF2 is hydrolyzed Large 60S rRNA subunit 5) associates with complex This releases all initiation factors from the complex Anticodon of initiator tRNA bound to AUG start codon in P site of ribosome Final Ribosome Assembled  Translation Structure of Ribosome Ribosomes have three sites for tRNAs: A (aminoacyl) site P (peptidyl) site E (exit) site. tRNA go from A -> P -> E mRNA is in a narrow groove of small ribosomal subunit Adjacent to A, P and E sites  Translation Elongation Elongation: Process of adding subsequent amino acids to the growing polypeptide chain Approximately ten amino acids added per second per ribosome Process: Step 1: Second aminoacyl-tRNA combines with elongation factor eEF1α-GTP 1) Step 2: Aminoacyl-tRNA is placed within the A site of the ribosome GTP is hydrolyzed and eEF1α -GDP is released Only tRNA with correct anticodon will trigger 2) conformational changes within the ribosome  Translation Elongation Elongation: Step 3: Amine nitrogen of AA on A site tRNA carries out nucleophilic attack on the carbonyl carbon of the AA at the P site tRNA Peptidyl transferase (enzymatic activity from the RNA (rRNA) component of the 60S. This catalytic activity is a feature of the rRNA itself) catalyzes the formation of peptide bond no energy is required. 3)  Translation Elongation Elongation: Step 4: Translocation Small rRNA subunit ratchets relative to large subunit Moving ribosome 3 nucleotides (1 codon) along the mRNA in the 5`-3` direction. dipeptidyl-tRNA in A site to P site deacylated tRNA in P site to E site driven by conformational changes in eEF2 following hydrolysis of bound GTP Step 5: deacetylated tRNA leaves ribosome, emptying E site 4) Step 6: elongation → 6) repeat of Steps 1-5 5)  Translation Termination: termination occurs at one of the 3 stop codons UAA, UAG, UGA no tRNA have anticodons complementary to a stop codon requires release factors, which recognize stop codons eRF1 and eRF3 → work together and recognize all the stop codons Step 1: eRF1 tri-peptide interacts with stop codon in A site Step 2: ester bond linking the nascent polypeptide to the tRNA is hydrolyzed Step 3: hydrolysis of eRF3-GTP releases eRF1 from A site Step 4: release of deacylated tRNA from P site, dissociation of mRNA from ribosome, disassembly of ribosomal subunits Requires several protein factors  Translation Polyribosomes A polyribosome (polysome) complex of multiple ribosomes on mRNA, allowing simultaneous translation increase the rate of protein synthesis Questions?