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Related Concept Videos

Transcription Initiation01:47

Transcription Initiation

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Initiation is the first step of transcription in eukaryotes. Prokaryotic RNA Polymerase (RNAP) can bind to the template DNA and start transcribing. On the other hand, transcription in eukaryotes requires additional proteins, called transcription factors, to first bind to the promoter region in the DNA template. This binding helps recruit the specific RNAP that can assemble on the DNA and start transcription.
The promoters and enhancers and their accessory proteins allow tight regulation of...
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Translesion DNA Polymerases02:10

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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Eukaryotic RNA Polymerases00:58

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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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Chromatin Structure Regulates pre-mRNA Processing02:41

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In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
The chromatin structure, especially...
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Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
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Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.

Masahiko Imashimizu1, Hiroki Takahashi2, Taku Oshima3

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RNA polymerase (RNAP) pausing in E. coli is caused by G-dC base pairs interfering with translocation and CpG sequences inducing errors. Gre factors resolve these transcriptional roadblocks.

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Measuring the Kinetics of mRNA Transcription in Single Living Cells
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Transcription elongation is often paused by DNA signals, impacting gene expression.
  • Transcription errors can cause prolonged pausing, potentially destabilizing the genome by hindering DNA replication.
  • In vivo mechanisms of pausing due to translocation blocks and misincorporation remain unclear.

Purpose of the Study:

  • To investigate RNA polymerase (RNAP) pausing patterns in Escherichia coli using a novel combined sequencing approach.
  • To elucidate the molecular mechanisms underlying RNAP pausing in vivo.

Main Methods:

  • Utilized native elongating transcript sequencing (NET-seq) combined with RNase footprinting of transcripts (RNET-seq).
  • Analyzed pausing patterns in Escherichia coli.

Main Results:

  • Identified that a G-dC base pair at the 5' end of the RNA-DNA hybrid impedes RNAP translocation, causing pausing in multiple RNAP states.
  • Demonstrated that CpG sequences in template DNA induce pausing and G-to-A errors, leading to RNAP backtracking.
  • Showed that Gre factors effectively proofread errors and rescue backtracked RNAP complexes.
  • Observed enrichment of pausing in 5' UTRs and antisense transcripts, with reduction in rRNA genes.

Conclusions:

  • Robust transcriptional pausing in E. coli involves RNAP interaction with G-dC at the RNA-DNA hybrid's upstream end, hindering translocation.
  • CpG DNA sequences are identified as inducers of transcriptional pausing and G-to-A errors.