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Transcription

Transcription. RNA Polymerase ‘reads’ template DNA strand to make complement RNA (mRNA code for a polypeptide). Associated with a gene(s) is an up-gene promoter/operator sequence for RNA polymerase binding and a down-gene termination sequence.

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  1. Transcription RNA Polymerase ‘reads’ template DNA strand to make complement RNA (mRNA code for a polypeptide). Associated with a gene(s) is an up-gene promoter/operator sequence for RNA polymerase binding and a down-gene termination sequence. Gene transcription can be regulated (on/off switch) negatively or positively by regulatory proteins (more later). Translation • Ribosome moves along the mRNA, codon-by-codon, adding appropriate amino acids via specific tRNAs to its growing polypeptide product.

  2. Transcription Genes + p/operator = operon. Gene(s) 3’ 5’ 5’ antisense 3’ sense Operator region Sigma-factor Antisense (template) strand Sense (complementary) strand Trailer sequence

  3. Promoter Region • Site for RNA Polymerase recognition and binding. • Recognition requires involvement of a sigma factor. • Different promoter types have their own sigma factor. • There can be an adjacent regulator binding site (= operator). Pribnow Box

  4. mRNA Elongation(recall that Us replace Ts) 5’ 3’ 5’ 3’

  5. Transcription Termination • Rho – dependent: a protein called “ρ” unwinds the DNA template – mRNA complex; thus weakening the association until mRNA and RNA polymerase falls off DNA. • Rho-Independent: here two G+C regions rich slow RNA polymerase; a hairpin forms in the mRNA weakening the mRNA-DNA template association; which is further weakened by poly-A stretch.

  6. rRNA and tRNA Products • rrn-gene has multiple rRNA & tRNA products. • Spacer RNA is spliced out by special nucleases. • rrn-operons are often located close to Ori. • Why might this have been selected? (think fast growth)?

  7. mRNA product • Within the leader is the Shine-Dalgarno sequence (5’AGGA3’). It complements a site on 16SrRNA of ribosome; used to bind a ribosome to mRNA for translation. • Trailer at 3’ end of mRNA was required for proper ending of transcription. • Translation of mRNA information into protein starts at the initiation codon (5’AUG3’); proceeds codon by codon until the end of gene, as identified by a stop, or non-sense, codon. • Coding region of mRNA can be polygenic (multiple genes = multiple starts / stops). Coding Region Direction of Translation

  8. Genetic Code: (3 nonsense;61 coding yet fewer tRNA) start

  9. Genetic code degeneracy; more than one codon for some amino acids. Results from tRNA “wobble” of first anticodon position; sometimes a novel nucleotide like inosine (e.g. ICC anticodon for Gly codons GGA, GGC, GGU). Codon written in reverse (3’→5’) 3’-A U G-5’

  10. Initiation Codon establishes the proper “reading-frame”!

  11. Translation

  12. Translation Initiation Three initiation factor proteins are involved (IF1, IF2, IF3). 30S subunit alone recognizes Shine-Delgarno sequence of mRNA, which binds initiation tRNA, IF1 & IF2; IF3 released. 50S subunit binds 30S (=70S), sandwiching mRNA and placing initiation tRNA at the P-site. A-site ready to receive tRNA for next codon. Ribosomes slide along mRNA in 5’→3’ direction.

  13. Translation Elongation Peptidyl transferase

  14. Translation Termination Stop codon Elongation of polypeptide continues until a stop codon is reached at the A-site. Ribosome stalls, allowing release factor proteins to bind. Upon dissociation of 50S from 30S, the IF3 binds 30S to make it receptive to a new mRNA start.

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