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.
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
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).
mRNA Elongation(recall that Us replace Ts)
5’
3’
5’
3’
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.
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)?
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
Genetic Code: (3 nonsense; 61 coding yet fewer tRNA)
start
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’)
Initiation Codon establishes the proper “reading-frame”!
Translation
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.
Translation ElongationPeptidyl
transferase
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.