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

3. Promoter Region Pribnow Box 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).

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). Direction of Translation Coding Region

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). 3’-A U G-5’ Codon written in reverse (3’→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.