9. Consider a prokaryotic gene and a eukaryotic gene. What are the differences i
ID: 300896 • Letter: 9
Question
9. Consider a prokaryotic gene and a eukaryotic gene. What are the differences in in the promoter sequences, the coding sequence, and the terminator sequences and their consequences for transcription? Suppose you want to put a gene fro coli and have E. coli produce the functional human protein. How must you modify the DNA sequence of interest for E. coli to be able to do this? Consider that transcription factors and RNA polymerase have to target the promoter for transcription to begin. Consider the way bacteria terminate transcription. Consider the requirement for RNA processing in eukaryotes. m humans in E. Daalatin of aene exnression:
Explanation / Answer
Difference in promoter sequences of prokaryotes and eukaryotes are :
Prokaryotic promoters contain two sequence motifs -10 and -35 elements located upstream of transcription start site. RNA polymerase binds slectively to promoter via interaction with sigma subunit. Sigma factor domain 2 binds to -10 element and domain 4 binds to -35 element of non template DNA strand. Some have an additional element called UP element, upstream of their -35 region. UP element is found in strong promoter like those of ribosomal RNA. Carboxy terminal domain of alpha subunit of RNA polymerase binds to this element. Some promoter even lack -35 element but have an extended -10 element. .
Eukaryotic promoters are complex and contain more than 2 sequence motifs which include TATA box, INR box, BRE, CCAAT and GC box. Proper assembly of RNA polymerase on promoter requires aid of large number of transcription factor than prokaryotes. There are 3 different RNA polymerases in eukaryotes which have different types of promoter compared to only one in prokaryotes. Majority of RNA polymerase III promoters extend into the sequences downstream of the transcription start site. In RNA polymerase I promoter upstream promoter element in addition of core promoter elements are found . core promoter is located at -45 to +20 and is sufficient for basal level transcription of genes. Polymerase II promoter have core elements(+40 to -50) , proximal elements ( -50 to -200 bp) and distal region (-200bp and upstream) . however a minimal Pol II promoter has only two elements i.e two elements re enough for basal level trancription. The core element consists of TATA box, BRE,DPE and INR. DPE is downstream promoter element located below start site, INR is initiator sequence present at the start site Five general transcription factors are required for proper assembly of Pol II to the promoter.
Difference in coding sequence is that eukaryotes contain monocistronic mRNA which codes for single protein. Prokaryotes have polycistronic mRNA which contain multiple different open reading frames (ORF) separated by intercisteronic region, each ORF codes for a different protein. For example lac operon genes are polycistronic. Eukaryotic mRNA contains exon and introns, introns are non-coding region present between exons (coding region) . Introns are removed and exons are joined before translation of mRNA. Prokaryotes do not contain introns.
Terminator sequences
Termination bacteria can be rho dependent or via intrinsic terminators. Instrinsic terminators are Stem loop structure having GC rich hairpin9 7-20 bp long) followed by a stretch of multipe Uracil. rho dependent termination sequences are bipartite and 200 nucleotide long. They are no loop structures. They contain rut sites where rho protein binds. Absence of a translating ribosome allows rho to bind the rut site and terminate transcription.
In eukaryotes Pol I termination sequences contain T1 and T2 sequences which bind to certain protein leading to bending. when pol I encounters the protein it back tracks leading to decreased DNA-RNA hybrid stability and termination of transcription.
In Pol II, termination and addition of poly A tail takes place simultaneously. The recognition signal is
5'--------------AAUAAA- 10-30 nucleotide-CA-----less than 30 nt-----GUrich----------- 3'
Cleavage and polyadenylation specificity factor ( CPSF) binds to AAUAAA (poly A signal sequence), cleavage stimulation factor (CstF) binds to GU rich region. An endonuclease then cleaves the mRNA molecule at CA site on which poly A polymerase can attach adenines. It adds approximately 200 adenine residues.
E.coli being a prokaryotes lacks introns and machinery required for its removal. Human genes contain a lot of introns which have to removed prior to its introduction into E.coli cells. For this mRNA molecules should be isolated from human cells and reverse trancribed to cDNA . This cDNA lacks any introns. cDNA molecule can be ligated to a plasmid vector downstream of a promoter. Exampe of vector is peT vector . In this gene is placed under T7 promoter, T7 polymerasec gene is present in the bacterial chromosome. vector contains ampicillin resistance gene. Codon bias also needs to be eliminated. codon bias is a phenomenon in which codon are non-randomly used between eukaryotes and proakryotes ,For example AGA ,AGG codons for arginine are very rarely used in bacteria, they use other codon CGA and CGG for arginine. This problem can be overcome by site directed mutagenesis in human cDNA molecule to replace rare codons with frequently used ones. Another strategy is to use special strains . These strains supply tRNA for the rarely codon in bacteria . Example rosetta (DE3) derived from BL-21 contains genes for AGG/AGA (Arg), AUA (Ile), CUA (Leu), CCC (Pro) and GGA (Gly) on a plasmid.
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