5- Desire for DNA - replication and transcription

why do we need DNA?

• RNA mutates spontaneously- the deamination of cytosine into uracil is very common, and hard to detect because it is single-stranded and because U is one of the bases anyway (unlike in DNA)

• it also has the 2’OH group, which allows it to form more H bonds and fold

• RNA itself is generally stable, but is unstable in the current protein world because of the presence of RNAases

• we need long stable sequences of information to produce enough proteins

how are the DNA building blocks produced?

• nucleoside diphosphates (NDPs) are converted into deoxynucleoside triphosphates (dNTPs) by ribonucleotide reductase (RNR), which removes the 2’OH, and kinase enzymes which add a third phosphate group

how does DNA replication occur?

• DNA helicase separates the two strands by breaking the H bonds

• complementary RNA primers are attached by primase at the start of the leading strand (replicated 5’ → 3’) and at regular intervals in the lagging strand (3’ → 5’)

• DNA polymerase adds on the dNTPs following base pairing rules (condensation reaction releasing pyrophosphate), producing Okazaki fragments in the lagging strand

• RNAse H degrades the RNA primers, and the fragments are extended until ligase joins the phosphate backbones together

what happens at the end of DNA replication in the lagging strand in eukaryotes?

• to form the final Okazaki fragment, telomerase extends the parental strand using an RNA template

• primase attaches an RNA primer to this extended DNA strand

• DNA polymerase extends the primer until the strands can be connected by DNA ligase

how is transcription initiated in prokaryotes?

• the sigma factor (a cofactor of RNA polymerase) recognises and binds to the Pribnow and TATA box motifs in the promoter region upstream of the initiation site

• this recruits RNA polymerase to bind to the DNA, produce a transcription bubble and begin RNA synthesis in the 5’ to 3’ direction using NTPs

• the sigma factor dissociates

how is transcription terminated in prokaryotes?

• after the stop codon is transcribed, termination signals found in the 3’UTR of the mRNA strand are also transcribed

• these can either be:

  • inverted repeats, which cause hairpin loops (through base pairing) that will terminate transcription by RNA polymerase

  • a rut termination sequence, which is recognised by the rho protein that binds to RNA polymerase, terminating transcription

how is transcription initiated in eukaryotes?

• the TATA binding protein (TBP), a subunit of transcription factor IID (TFIID), binds to the TATA box in the promoter region upstream of the initiation site

• this recruits multiple proteins, including RNA polymerase II (Pol-II), which forms a transcription bubble, detaches from TFIID and begins to transcribe the RNA upon phosphorylation using NTPs

how is transcription terminated in eukaryotes?

• after the stop codon is transcribed, a polyA signal (AAAUAAA) is found in the 3’UTR

• this causes cleavage downstream by endonuclease enzymes, terminating transcription

• a polyA tail (200-250 A nucleotides) is added to the 3’ end to increase the mRNA stability

• the introns are then removed from this preRNA by splicing, catalysed by spliceosome (a ribozyme)

how is mRNA modified differently in prokaryotes and eukaryotes and why?

• prokaryotes don’t have membrane-bound nuclei, so transcription and translation can occur simultaneously

  • at the 5’ end, they just have a triphosphate purine nucleotide

• eukaryotic mRNA has to be transported out of the nucleus, so it is modified in a more complicated way:

  • a 5’ cap is added (made from guanosine triphosphate, and involving the methylation of the first two bases)

  • at the 3’ end a polyA tail is added (200-250 A nucleotides)

  • introns are also removed by splicing