Macromolecular Synthesis-Transcription and Translation Flashcards
Transcription
- bacteria regulate protein synthesis at level of Tc
- mRNA in bacteria is generally unstable
- RNA pol can initiate new strand synthesis
Architecture of a gene
Coding strand-the one that looks like the mRNA sequence (has the codon sequence)
Template strand-the one that is read
Tc Initiation (promoter and RNA pol)
- 10 and -35 are promoter region
- RNA pol (holoenzyme) consists of 5 subunits; alpha (2), beta, beta1, sigma, omega-6 proteins
- core pol can synthesize RNA, core is w/o sigma factor
- sigma helps RNA pol recognize the promoter (specificity)
Sigma Factors
- sigma 70=housekeeping
- alternate sigma factors recognize different promoter sequences
- sigma54 is N related, different structure
- sigma 32 turns on heat shock genes during stress
Promoter Strength
-closer to the consensus sequence, the stronger the promoter
-spacing b/w -10 and -35 is impt
-sequence of spacer region is sometimes impt
-control frequency and strength of pol binding
TTGACA–17–TATAAT=consensus promoter
Tc Initiation
- polymerase binds promoter-closed complex
- DNA unwinds to form open complex
- holoenzyme can form open complex
- ribonucleotides are incorporated beginning form Tc start site, not start codon
Elongation
- sigma subunit dissociates
- DNA-RNA hybrid of 18 nt as polymerase synthesizes the mRNA–>Tc bubble
Termination-Intrinsic terminators (factor independent)
- secondary structure in newly made message that results in pol dissociation
- inverted repeats form hairpin
- unfavourable for pol to bind to
Termination-Rho-dependent Terminators
- rho binds to rut site on newly made message and moves up towards pol-DNA complex
- pol reaches a rho-dependent termination sequence and stalls
- rho catches up (through ATP hydrolysis) and releases RNA from elongation complex
- rho acts together with sequence that stalls the polymerase
Operons
- genes encoding for proteins w/ related functions are often clustered together into operons (eg. lactose operon contains genes involved in lactose metabolism)
- one promoter is used to make a mRNA that will be translated into multiple proteins
- polycistronic
Translation
- ribosomes assemble on the mRNA
- recruit charged tRNAs and from peptide bonds b/w the aa’s that the tRNAs bring
- in bacteria Tc and TL are coupled
Ribosomes
-made of RNA and proteins
-small subunit binds mRNA
-large subunit provides enzymes needed to form peptide bonds
-binds 5’ end of mRNA, scans until it gets to 1st AUG
2 sites for charged tRNAs
-P (peptidyl) site
-A (aminoacyl) site
Translation Initiation
- codons are paired w/ anticodon regions of tRNAs
- shine delgarno sequence (ribosome binding site) is recognized by the 16s RNA in small subunit of the ribosome (30s)
Degeneracy of the Genetic Code
- multiple codons for the same aa
- buffers the genome against mutations
- changes in the 1st and 2nd position can cause mutations
Aminoacyl-tRNA Synthetases (AARSs)
- attach the correct aa to the tRNA
- 1st aa is formulated-methionine (f-Met)
Translocation
- peptide bond is formed b/w tRNA at A site and peptidyl-tRNA in P site
- uncharged tRNA leaves P site
- ribosome moves along the message to the new A site
- need GTP
TL Termination-release factors
RF 1-recognizes UAG, UAA
RF2-recognizes UGA, UAA
-release factors bind stop codons
-dislodge growing polypeptide chain, ribosome disassembles
Common Themes in Macromolecular Synthesis
- Initiation-protein machines recruited by specific sequences in the nucleic acid
- Elongation
- Termination-specific sites on nucleic acids act to disassemble the protein machines, often by recruiting specific proteins (Tus, Rho)
