Day 2- Cellular Processes in Single Cells II: Transcription

Good morning everyone! The sky has cleared up a bit today (with a bit drizzles). After the first day of ideas and the Shilin night market tour, we are all ready for another day of interesting sciences!

Achillefs Kapanidis- Using tracking PALM to study bacterial transcription and chromosome organization in live bacterial cells

In order to understand RNA polymerase (RNAP) behavior and its role in nucleoid organization in vivo, Achillefs’ team used photo-activated localization microscopy single-molecule tracking, and they were able to tell apart diffusing RNAP from those that are bound to DNA.

They found that RNAP has periphery bias that is dependent on active transcription, and RNAP clustering is a function of growth. These RNAP clusters are having a similar mobility as DNA. Interestingly, when they image the nucleoid and RNAP using 3D structural illumination, they found that RNAP will form large clusters at regions of low DNA density in rich media, leading to the suppression of other genes. The mechanism they proposed is that RNAP redistribution is due to changes in gene expression, such as stress, mutation, and overexpression. Moreover, they also found that RNAP will interact with non-specific DNA substantially.

They are also developing assays to study the non-specific interactions of DNA binding proteins with chromosomal DNA, which would definitely be a useful tool for this field!

 

Nam Ki Lee- Direct observation of transcription in a living bacterial cell

Nam Ki and his team are studying the coupling of transcription and translation, and how these two spatially separated but functionally related processes are cooperatively regulating the movement and the effective expression of genes.

They observed the movement of the actively transcribing T7 RNAP toward outside nucleoid, and this was affected by the translation by ribosome. Furthermore, they found that the movement of genes by transcription-translation coupling is seen in both E. coli RNAP and T7 RNAP. They also measured the in vivo kinetics of the T7 RNAP transcription on-rate and elongation rate, and found that deletion of the ribosomal binding site doesn’t change the elongation rate, but enhanced the transcription on-rate 1.7-fold, indicating a close relationship of transcription-translation effect.

The model that they propose is that transcription starts within the nucleoid, and then the DNA-RNAP-ribosome complex will move to the outside of the nucleoid, and the transcription initiation is enhanced!

 

David Rueda- Imaging Small Cellular RNAs with Fluorescent Mango RNA Aptamers

David and his group set out to develop fluorogenic RNA aptamers that has improved physicochemical properties (i.e., thermal stability, fluorescence brightness and ligand affinity) and better signal-to-noise ratio, and they developed “mango” I-IV (after spinach, lots of veggies and fruits *laugh*).  Interestingly, mango IV is resistant to formaldehyde fixing, which is particularly useful for cell fixation.

They shown that these aptamers could be used to image small non-coding RNAs (such as 5S rRNA and U6 snRNA) in both live and fixed human cells with improved sensitivity and resolution. In the 5S rRNA example, they showed that 5S rRNA foci are not processing bodies, but instead are associated with mitochondria!  They were also able to image U6 snRNA in live cells, and they found that there are 3 types of behavior, no moving, low mobility, and high mobility.

Interestingly, they referred to the idea of Bo Huang and developed CRISPR-mango for imaging of telomeres and specific loci! This would definitely open up a new world of RNA imaging in cells!

 

Xiaoli Weng- Using Superresolution Fluorescence Microscopy to probe the spatial organization of transcription in E. coli

Xiaoli is trying to gain insight into the regulation of gene expression at the global level by using superresolution fluorescence microscopy.

They found that RNAP forms clustered distribution under fast growth, and globally stopping transcriptions has the largest perturbations. They also probed the colocalization of the elongation factor NusA and RNAP, and found they are together in elongation complexes, with nascent rRNA. Interestingly, when they perturbed rRNA transcription with serine hydroxamate treatment or in rrn deletion strain, they found that certain RNAP independent of rRNA synthesis are retained.

Therefore, the formation of RNAP clusters and active rRNA synthesis could be independent, and genes could potentially localize with RNAP clusters to have better regulation and more efficient transcription.

 

Ivy Pei-Tzu Huang (Howard Lab & Xiong Lab, Yale University)

 

 

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