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TranscriptionComments

Transcription

In transcription, a complementary RNA sequence is made from a strand of DNA. In other words, transcription is the transfer of genetic information from DNA to RNA.

Transcription is the beginning of the process that ultimately leads to a protein or peptide. Transcription is performed by RNA polymerase (RNAP). Transcription has less and less effective proofreading than in replication.

In other ways, tTranscription is similar to replication: it is antiparallel, 5'-3' & there is complementary alignment of strands.

Transcription has 3 stages: initiation, elongation and termination. The universal substrates are a DNA template and NTPs (nucleoside triphosphates, instead of dNTPs in replication).

To find the promoter, if you take a bunch of E. coli promoters and line them all the way up, count back so you can begin to see the conversation, and that was the first designation of -10 and 35. Up elements increase the strength of a promoter and the likelihood that transcription will occur. mRNA can be read in different ways. We have the ribosome binding site (RBS). The 5' of the RNA is depicted a concensus sequence, the concensus sequence of a start codon is AUG.

The Cap is recognized in the cytoplasm by special initiation factors to allow assembly of a ribosome at the mRNA initiation site. Because the initiation of translation involves Cap recognition, each protein in eukaryotic cells is encoded on its own mRNA. The capped base of an mRNA corresponds to the start site of transcription of the precursor RNA from which it is processed.

Eukaryotic transcription has 3 RNAPs (RNAPI, RNAPII & RNAPIII) while prokaryotic transcription has only one. In eukaryotes, there are many general transcription factors (GTFs) while in prokaryotes σ is the only GTF.

Prokaryotic transcription

Initiation

RNA Polymerase (RNAP) recognizes and binds to promoter region on dsDNA, forming the closed complex.

Around the initiation site (+1), the DNA is unwound & becomes single-stranded; the RNAP/ssDNA structure is the open complex. The RNAP transcribes the DNA, but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNAP because the exit channel is blocked by the σ-factor. The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.

Most transcripts originate utilizing adenosine-5'-triphosphate (ATP), GTP being used less often at the +1 site. UTP and CTP (pyrimidines) are disfavored.

The σ-subunit binds to the promoter region, then the core polymerase binds to it (a holoenzyme is a core-polymerase-σ-subunit complex). The promoter refers to the start point of transcription. Since transcription starts at +1, the promoter is between -35 and -10. The start site is relative to the promoter region. There is no primer. Three stop codons: UGA, UAG, UAA
RNAP does everything, including helicase activity by unwinding closed compled DNA. Then the RNAP starts unwinding it into an open complex.
  • Elongation, you just keep adding nucleotides to your mRNA.
  • Elongation

    The RNAP runs along the DNA, synthesizing the complementary RNA in the process. In prokaryotes, the nascent mRNA is translated co-transcriptionally by ribosomes. Some proofreading occurs during this process: Pyrophosphorolytic editing - RNAP immediately removes incorrect pairs reversing the reaction that put them together. Hydrolytic editing - RNAP backtracks one or more bases to remove an incorrect pair, stimulated by Gre factors. In prokaryotes, this occurs in the cytoplasm which is why translation occurs at the same time.
    σ Factor FateIt is not favorable to have σ-factor on holoenzyme since core polymerase does not want to associate it. The sigma factor falls away before elongation. Therefore, the core polymerase performs elongation (not the holoenzyme).

    Termination

    Termination has two mechanisms: intrinsic (ρ-independent) & ρ-dependent. In intrinsic termination, a terminator sequence within RNA signal RNAP to stop. Terminator sequence is palindromic, & forms a stem-loop hairpin structure leading to dissociation of RNAP from DNA template. In rho-dependent terminatio, ρ binds & runs along the mRNA toward the RNAP. When they collide, it causes RNAP to dissociate from DNA (terminating transcription). ρ stops RNA synthesis at specific sites.

    • Rho dependent termination: rho uses it's helicase activity to separate mRNA from DNA
    • Rho-independent termination:
      • loop formed
      • hairpin loop formed due to Gs and Cs wanting to bind together
      • That association makes DNA-RNA hybrid unwind
      • Due to weaknesses of DNA-RNA hybrids, and weak AU base pairs, hybrid falls apart

    Eukaryotic transcription

    Eukaryotes have 3 RNAPs:

    • RNAPI makes rRNA
    • RNAPII makes mRNA
    • RNAPIII makes tRNA & small RNAs

    Eukaryotes have 5 GTFs

    • Tata Binding Protein (TBP)
    • TFIIH
    • FT TFIID
    • TFIIB
    • TFIIH

    Eukaryotic promoter has 4 parts

    • B Recognition Element
    • TATA box
    • Initiator
    • Downstream promoter element

    Initiation

    TBP recognizes and binds to the tatabox (-31 to -26). TBP is the σ of eukaryotes. Once bound, TBP recruits the other factors.
    TFIIH, a helicase, makes the DNA go from a closed to open complex. TFIIH phosphorylates the carboxy terminal domain of RNAP, causing a conformational change in RNAP which results in the factors being released (hydroxyl groups are phosphorylated; threonine, serine and tyrosine have 3 of them; serine and tyrosine are predominant on the carboxy terminal domain). TFIIH is a large General Transcription Factor (GTF) of 9 subunits, nearly as large as Pol II. Two of the subunits are homologous to DNA helicases, enzymes that use energy from ATP hydrolysis to separate the strands of a DNA double helix. One of the subunits is a kinase that phosphorylates Ser5 of the Pol II large subunit C-terminal domain (CTD) heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

    The initial complex containing properly distorted DNA becomes the platform for assembly. TFIIB interacts specifically with TBP, and the promoter. Usually, RNAPII is preloaded with a variety of factors. As factors load onto this sort of open complex, a helicase protein is required to make this an open complex. This complex is tied to the promoter & keeps trying to commence transcription, but it is unable to go. It starts & makes short abortive-initiation transcripts about 10 base pairs long because it is ready to go but cannot. It has mousetail, the C-terminal domain tail is phosphorylated & the initiation factors are shed so that RNAPII remains on the RNA & can go beyond abortive initiation. Whether or not and how you phosphorylate this tail depends on the speed RNAP II CTD has YSPTSPS heptapeptide repeats. Each has sites for specific serine kinases (1 in TFIIH).

    Phosphorylation enables the complex to shed initiation factors and proceed to elongation. In eukaryotes, transcription takes place in the nucleus (where the DNA is). This allows for the temporal regulation of gene expression via sequestration of RNA in nucleus, and allows for selective transport of RNAs to the cytoplasm, where the ribosomes reside. Further complexity is added by the multitude of transcripton factors and signaling pathways that may interact in combination to mediate cell-type and developmental transcriptional regulation. Primary (initial) mRNA transcripts in eukaryotic cells are synthesized as larger precursor RNAs that are processed by splicing out introns (non-coding sequences) and ligating exons (non-contiguous coding sequences) into the mRNA. Primary transcripts for some genes can be large.