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Transcript & Transla
| Question | Answer |
|---|---|
| How Does Transcription Start at the Correct Site (Initiation)? | Sigma subunit recognizes specific sequences in the DNA. Different sigma factors recognize different promoters. |
| Sigma 70 (σ⁷⁰): | Used during initiation, is responsible for recognizing most genes under normal conditions. |
| What does Sigma 70 (σ⁷⁰) bind to? | Pribnox Box TATAAT sequence at -10, -35 region TTGACA sequence |
| What determines how often transcription will start at a site? | RNA Polymerase binding determines how often transcription will start at a site. The start site is an A base. Transcrip happens in 5’ to 3’. Sigma sub is required to start transcription, then dissociates from RNA polymerase. |
| Strong promoters | frequent transcription initiation |
| Weak promoters | infrequent transcription |
| RNA Polymerase Process (Transcription Elongation): | 1) RNA polymerase unwinds DNA ahead and rewinds it behind, 2) synthesizes RNA 5' -> 3' 3) reads the DNA template 4) produces RNA identical to DNA except U replaces T, 5) forms a RNA:DNA helix in bubble, 6) RNA polymerases can follow same template |
| How is Transcription Terminated (Method 1): | by a special termination protein called Rho (p), binds to newly formed RNA and moves down the RNA towards RNA polymerase, RNA polymerase pauses at Rho termination sites, Rho catches up, unwinds RNA:DNA hybrid, RNA polymerase dissociates |
| Main Idea of Rho: | Rho (p) acts like a helicase |
| How is Transcription Terminated (Method 2): | 1 RNA forms stem-loop (hairpin) structure, Strong GC base pairs in stem, Palindromic sequence required 2 Followed by string of U residues (~6-8) 3 AU base pairs are very weak 4 Hairpin + weak AU pairs destabilize complex 5 RNA polymerase dissociates |
| rRNA Processing: | In E. coli, rRNAs are made as one long RNA, sometimes with tRNAs. RNases (like RNase III and RNase E) then cut it into separate rRNAs for ribosome assembly. |
| tRNA Processing: | Involves trimming extra sequences from the ends of tRNA, adding a CCA tail if needed, and modifying certain bases to make a mature, functional tRNA. |
| Polycistronic mRNA: | mRNA transcribed from multiple genes (operon), Contains multiple ORFs (Open Reading Frames), Gene = cistron = ORF (terms often used interchangeably) |
| Polycistronic mRNA Structure: | One promoter at front, One terminator at end. Each gene has its own Ribosome Binding Site (RBS) — Also called Shine-Dalgarno sequence. |
| Shine-Dalgarno sequence: | found in polycistronic mRNA, ensures correct AUG start codon recognition and allows differential translation of genes |
| Transcription in Archaea and Eukaryotes is More Complex: | eukaryotes have three RNA polymerases, prokaryotes have one RNA polymerase, Archaea have one RNA polymerase similar to RNA Polymerase II of eukaryotes. |
| Major Difference Between A, E, and B Transcription (Initiation): | Archaea and eukaryotes require transcription factors → TBP (TATA Binding Protein): Binds TATA sequence, TFB (Transcription Factor B): Binds BRE and INIT sequences, Transcription factors bind first, then recruit RNA polymerase. |
| If Eukaryotes and Archaea use Transcription Factors, what does bacteria use? | Bacteria use a sigma subunit (part of polymerase). |
| Sigma Subunit Vs. Transcription Factors: | ss are component of the bacterial RNA polymerase holoenzyme that binds to promoter regions to initiate transcription at start site, while transcript are proteins that regulate gene expression by either activating or repressing transcription. |
| Eukaryotic mRNA Processing (Three Major Processing Steps): | 5' Capping, 3' Polyadenylation, Splicing |
| 5' Capping (Eukaryotic mRNA Processing): | 7-methylguanosine cap added, Protects from degradation, Required for translation initiation |
| 3' Polyadenylation (Eukaryotic mRNA Processing): | Poly(A) tail (~200 A's) added, Protects from degradation, Aids in export from nucleus |
| Splicing (Eukaryotic mRNA Processing): | Introns (intervening sequences) removed, Exons (expressed sequences) joined together, Performed by spliceosome, Allows alternative splicing (multiple proteins from one gene) |
| Introns & Exons: | the former are intervening sequences and the latter are expressed sequences → not found in bacterial mRNAs! |
| Amino Acids: | 22 Amino Acids Total → 20 common amino acids, 2 rare: Selenocysteine (Sec, U), Pyrrolysine (Pyl, O). Amino acids are classified by R-group |
| Hydrophobic (Nonpolar), Amino Acid Classification: | Glycine, Alanine, Valine, Leucine |
| Polar (Uncharged), Amino Acid Classification: | Serine, Glutamine, Asparagine |
| Charged Amino Acid Classification: | Acidic (negative): Aspartate, Glutamate Basic (positive): Lysine, Arginine, Histidine |
| Peptide Bonds: | Link amino acids together, Form between carboxyl group of one amino acid and amino group of next |
| N-terminus (Peptide Bonds): | free amino group |
| C-terminus (Peptide Bonds): | free carboxyl group |
| Protein Structure Levels (Primary (1°) Structure): | Linear sequence of amino acids, Determined by gene sequence |
| Secondary (2°) Structure: | a-helix: Right-handed spiral, stabilized by H-bonds, β-sheet: Extended strands, H-bonds between strands (parallel or antiparallel) |
| Tertiary (3°) Structure: | Overall 3D shape of single polypeptide → Stabilized by Hydrogen bonds, Ionic interactions, Hydrophobic interactions, Disulfide bonds (S-S) between cysteine residues |
| Quaternary (4°) Structure: | Assembly of multiple polypeptides (subunits), Example: Hemoglobin (2α + 2β chains), Example: Insulin (A + B chains) |
| tRNA 2D Structure: | often drawn as a cloverleaf. Some bases modified after synthesis (like pseudouridine), acceptor stem is always CCA and is added to the 3’end after the tRNA is transcribed, anticodon is specific for each amino |
| Charging of tRNAs — addition of amino acid (ARS Enzyme): | ARS enzyme recognizes different parts of the tRNA and adds the correct amino acid acceptor to acceptor stem, there is a different ARS enzyme for each amino acid combination, the reaction requires ATP and releases AMP plus pyrophosphate |
| ARS Two Enzymatic Steps: | 1) activating the amino acid by adding AMP and then transferring the amino acid to tRNA, 2) the carboxyl end of the amino acid is attached to the 3’ end of the tRNA |
| The Genetic Code (Codon Properties): | 64 possible codons (4³ = 64), Code for 20 common amino acids + start/stop signals, Triplet code: Each codon is 3 nucleotides |
| Why is the Genetic Code Degenerate? | Because several amino acids can be encoded by more than one codon (example: six codons for leucine). |
| Start Codon: | AUG is the start codon most of the time. |
| Stop codons (nonsense codons): | UAA, UAG, UGA. Do not code for amino acids. Signal termination. |
| Wobble Hypothesis: | degenerate codons for the same amino acid often differ in the 3rd position on the mRNA. This can often form a mismatched base pair. |
| Example of the Wobble Hypothesis: | there are two codons for lysine: AAA and AAG. But E.coli does NOT have a tRNA for the AAG codon. E.coli only has a lysine tRNA with the anticodon UUU. |
| Wobble Rules: | G can pair with U or C, I (inosine) can pair with U, C, or A, Reduces number of tRNAs needed (~45 instead of 61). |
| Reading Frames in mRNA: | Three possible reading frames, Start from different positions (nucleotide 1, 2, or 3), Only one is correct (determined by start codon and RBS) |
| Reading Frames in DNA: | Six possible reading frames, Three on each strand (DNA is double-stranded) |
| Open Reading Frame (ORF): | a start codon followed by a number of amino acids encoding codons ending with a stop codon. |
| Translation Initiation in Bacteria (Ribosome Structure): | The 30S subunit binds mRNA and the initiator tRNA at the start codon, then the 50S subunit joins to form the complete 70S ribosome ready for protein synthesis. |
| The Translation Initiation Ribosome Has Three Major Sites: | Exit, Peptide, and Acceptor |
| E site (Exit): | Where deacylated tRNA exits |
| P site (Peptide): | Holds tRNA with growing polypeptide chain |
| A site (Acceptor/Aminoacyl): | Where new aminoacyl-tRNA enters |
| Bacterial Translation Initiation Steps: | The 30S subunit binds the mRNA’s Shine-Dalgarno sequence, the initiator tRNA-fMet attaches to the P site, the 50S subunit joins using GTP, forming the 70S ribosome ready for elongation. |
| Bacterial Translation Elongation Steps: | A charged tRNA binds to the A site, a peptide bond forms between the A- and P-site amino acids, the ribosome moves one codon (tRNAs shift A→P→E), and the empty tRNA exits from the E site. |
| Bacterial Translation Rate: | ~15-20 amino acids per second in bacteria |
| Ribosomes Read mRNA Similar to RNA Polymerase Reading DNA (one after the other): | RNA polymerases “line up” on the DNA to synthesize RNA. Meanwhile, ribosomes “line up” on the mRNA to synthesize proteins. |
| Bacterial Translation Termination: | Release Factors (RF) recognize stop codon, 70S ribosome dissociates into 30S and 50S, Components recycled for next round |
| Transcription & Translation Occur at the Same Time in Bacteria: | RNA polymerase reads DNA 3′→5′ and builds mRNA 5′→3′; ribosomes then read mRNA 5′→3′ to make proteins from the N-terminus to the C-terminus. |
Created by:
smurtab