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Lernmaterialien für Genex an der Universität Wien

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Gene expression steps?

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  1. Transcription control by proteins and ncRNAs: they decide when the genes are transcribed, in certain situations, certain cell types -> how that works?
  2. Transcription processing (Splicing, Transport in eukaryotic cells out of the nucleus) by proteins and snRNAs (small nuclear RNAs)
  3. Transcript stability (Transcripts can live from minutes to hours) controlled by proteins and ncRNAs (non-coding RNAs)
  4. Translation control by proteins and ncRNAs
  5. Post-translational modification (When proteins are made they can be in active or inactive states/conformation) converted by proteins (enzymes); attach a chemical modification like phosphate to the protein, and it decides if it’s active or not  (protein kinases and protein phosphatases)
  6. Protein stability by proteins (enzymes) proteins are turned over, resynthesized and degraded
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Phage Lambda uses antitermination proteins as switches between early and late phases of gene expression. How?

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  • Early phase: virus makes sure infection is established
  • Late phase: genome replication, capsid building.. 
  • There is only a promoter before the early phase genes, a terminator sequence in between the early and late phase genes, and no promoter for the late phase genes
  • Early region transcribed -> N protein is made -> makes sure that intermediate phase can be transcribed -> Q protein is made -> switch for late phase
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The codon sun

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Codon sun has a third-base degeneracy, which means that the third position has a lesser effect on the meaning of the codon as the first two. Chemically similar amino acids like Glu or Asp for example usually have related codons, so even if the third-base gets exchanged, it won’t make a difference in the overall structure and function of the protein. Third base degeneracy accelerates translation


Chemically similar amino acids often have related codons minimizing the effects of mutation.


Codon-Anticodon recognition involves wobbling. The pairing of the first place of the anticodon and third place of the codon can differ from the Watson-Crick pairings.


Every codon starting with CC will code for proline. It is a helix breaker protein so it is very important to only be inserted into the sequence when it is needed. In order to reduce the error, proline is encoded by all nucleotides in the third position after a CC.



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How do you determine if a transcription factor really has a role in transcription of a gene?

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  1. Co-transfection using a reporter gene with TF binding site in the promoter region and another plasmid carrying the gene for the TF
  2. Loss-of-function assays using siRNA mediated knock-down, or knock-outs by gene targeting
Lösung ausblenden
TESTE DEIN WISSEN

Phage-encoded σ-factors direct the switch from early to late gene expression in B. subtilis phage SPO1. How?

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TESTE DEIN WISSEN

This phage has to switch from early genes to late genes. It uses σ-factors to switch. The early promoter uses one sigma factor, then another sigma factor will be synthetized, holoenzymes will be formed with the second sigma factor, it finds intermediate genes and so on. 


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Two component systems?

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Two component systems comprise of a sensor that senses the change in environment and a response regulator which is activated by the sensor and in its activated form work as a transcriptional factor and turns on genes needed to react to the change. 


eg. phoR-phoB for intracellular phosphate levels


eg. NtrB-NtrC system glutamine synthase gene for oganic nitrogen levels

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Transactivating domains of transcriptional factors?

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Transactivating domains (TADs)

  • They do not really form stable secondary structures
  • Mediate protein interactions required for promoter activation, transcriptional initiation or transcriptional elongation
  • Poorly structured
  • Classified by predominance of aas with common chemical properties like Acid TADs, Gln-rich , Pro-Rich, Ser/Thr-Rich TADs…
  • Different classes of TADs may contact different protein complexes during promoter activation/transcriptional initiation
Lösung ausblenden
TESTE DEIN WISSEN

DNA Microarrays = gene chips?

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TESTE DEIN WISSEN

Good if I want to know ALL the genes that are active upon transfection or other stimulus

  • Simultaneous analysis of many genes or all genes in the genome!
  • Each spot contains a probe that will hybridize to one of the genes of the species for example (6400 in yeast)

You isolate RNA in different conditions (normal, infection/stress...), reverse transcribe RNA to cDNA so that one of the nucleotides is coupled to a fluorescent dye, different in the different conditions.

Then load it onto microarray chip, upon hybridization fluorescence can be measured. 

Reading results is based on the colors, if the gene transcribed in both conditions, the mixture of the two colors is visible. 


Outdated.

Lösung ausblenden
TESTE DEIN WISSEN

How do you find transcription factors / proteins that bind to your DNA sequence (promoter for example) ?

Lösung anzeigen
TESTE DEIN WISSEN

In vitro: EMSA (electrophoretic mobility shift assay)


To see if it's not just an in vitro artefact: ChiP (chromatin-immunoprecipitation) and ChiP-Seq


Or by bioinformatics in databases looking for transcription factor binding sites that match my DNA sequence

Lösung ausblenden
TESTE DEIN WISSEN

How do you determine if the increased level of mRNA upon stimulus is really due to transcriptional control?

Lösung anzeigen
TESTE DEIN WISSEN

Nuclear run on assay or Gro-Seq


Nuclear run on assay is less used today, mostly Gro-Seq is used.

Lösung ausblenden
TESTE DEIN WISSEN

GRO-Seq = genomic run on sequencing

Lösung anzeigen
TESTE DEIN WISSEN

With this method you can see if the increase in mRNA is due to transcriptional control.

This is very similar to the nuclear run on assay, only this one doesn't use radioactivity and uses RNA-Seq. Filtering is based on biotin-streptavidin interaction.


  • Isolate nuclei, elongate transcript in the presence of chemically modified UTP (e.g. bio-UTP)
  • Isolation of chromatin-associated, nascent mRNA using the chemical modification of U (e.g. with Streptavidin)
  • Reverse transcription -> RNA-Seq (not on total cellular RNA analysis but on nascent transcripts only! -> this corresponds to transcription rate)
  • Bioinformatic analysis of transcript frequency



Lösung ausblenden
TESTE DEIN WISSEN

Accuracy of tRNA synthetases

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TESTE DEIN WISSEN

Because of the similarity of some amino acids, finding the accurate one is a difficult task for the enzyme. Specificity thereby is controlled by proofreading reactions. After the amino acid has been linked to the AMP on the synthesis site and the tRNA binds to the enzyme, the enzyme runs a second amino acid proofreading step. It tries to force the amino acid into a second pocket, the editing site, which excludes the correct amino acid but is open for the similar ones. In the case that an incorrect amino acid is bound to the AMP or tRNA, this amino acid will be transferred to the editing site of the enzyme, where it will be removed by hydrolysis. The tRNA charging makes one mistake approximately in 40000 couplings.

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Q:

Gene expression steps?

A:
  1. Transcription control by proteins and ncRNAs: they decide when the genes are transcribed, in certain situations, certain cell types -> how that works?
  2. Transcription processing (Splicing, Transport in eukaryotic cells out of the nucleus) by proteins and snRNAs (small nuclear RNAs)
  3. Transcript stability (Transcripts can live from minutes to hours) controlled by proteins and ncRNAs (non-coding RNAs)
  4. Translation control by proteins and ncRNAs
  5. Post-translational modification (When proteins are made they can be in active or inactive states/conformation) converted by proteins (enzymes); attach a chemical modification like phosphate to the protein, and it decides if it’s active or not  (protein kinases and protein phosphatases)
  6. Protein stability by proteins (enzymes) proteins are turned over, resynthesized and degraded
Q:

Phage Lambda uses antitermination proteins as switches between early and late phases of gene expression. How?

A:
  • Early phase: virus makes sure infection is established
  • Late phase: genome replication, capsid building.. 
  • There is only a promoter before the early phase genes, a terminator sequence in between the early and late phase genes, and no promoter for the late phase genes
  • Early region transcribed -> N protein is made -> makes sure that intermediate phase can be transcribed -> Q protein is made -> switch for late phase
Q:

The codon sun

A:

Codon sun has a third-base degeneracy, which means that the third position has a lesser effect on the meaning of the codon as the first two. Chemically similar amino acids like Glu or Asp for example usually have related codons, so even if the third-base gets exchanged, it won’t make a difference in the overall structure and function of the protein. Third base degeneracy accelerates translation


Chemically similar amino acids often have related codons minimizing the effects of mutation.


Codon-Anticodon recognition involves wobbling. The pairing of the first place of the anticodon and third place of the codon can differ from the Watson-Crick pairings.


Every codon starting with CC will code for proline. It is a helix breaker protein so it is very important to only be inserted into the sequence when it is needed. In order to reduce the error, proline is encoded by all nucleotides in the third position after a CC.



Q:

How do you determine if a transcription factor really has a role in transcription of a gene?

A:
  1. Co-transfection using a reporter gene with TF binding site in the promoter region and another plasmid carrying the gene for the TF
  2. Loss-of-function assays using siRNA mediated knock-down, or knock-outs by gene targeting
Q:

Phage-encoded σ-factors direct the switch from early to late gene expression in B. subtilis phage SPO1. How?

A:

This phage has to switch from early genes to late genes. It uses σ-factors to switch. The early promoter uses one sigma factor, then another sigma factor will be synthetized, holoenzymes will be formed with the second sigma factor, it finds intermediate genes and so on. 


Mehr Karteikarten anzeigen
Q:

Two component systems?

A:

Two component systems comprise of a sensor that senses the change in environment and a response regulator which is activated by the sensor and in its activated form work as a transcriptional factor and turns on genes needed to react to the change. 


eg. phoR-phoB for intracellular phosphate levels


eg. NtrB-NtrC system glutamine synthase gene for oganic nitrogen levels

Q:

Transactivating domains of transcriptional factors?

A:

Transactivating domains (TADs)

  • They do not really form stable secondary structures
  • Mediate protein interactions required for promoter activation, transcriptional initiation or transcriptional elongation
  • Poorly structured
  • Classified by predominance of aas with common chemical properties like Acid TADs, Gln-rich , Pro-Rich, Ser/Thr-Rich TADs…
  • Different classes of TADs may contact different protein complexes during promoter activation/transcriptional initiation
Q:

DNA Microarrays = gene chips?

A:

Good if I want to know ALL the genes that are active upon transfection or other stimulus

  • Simultaneous analysis of many genes or all genes in the genome!
  • Each spot contains a probe that will hybridize to one of the genes of the species for example (6400 in yeast)

You isolate RNA in different conditions (normal, infection/stress...), reverse transcribe RNA to cDNA so that one of the nucleotides is coupled to a fluorescent dye, different in the different conditions.

Then load it onto microarray chip, upon hybridization fluorescence can be measured. 

Reading results is based on the colors, if the gene transcribed in both conditions, the mixture of the two colors is visible. 


Outdated.

Q:

How do you find transcription factors / proteins that bind to your DNA sequence (promoter for example) ?

A:

In vitro: EMSA (electrophoretic mobility shift assay)


To see if it's not just an in vitro artefact: ChiP (chromatin-immunoprecipitation) and ChiP-Seq


Or by bioinformatics in databases looking for transcription factor binding sites that match my DNA sequence

Q:

How do you determine if the increased level of mRNA upon stimulus is really due to transcriptional control?

A:

Nuclear run on assay or Gro-Seq


Nuclear run on assay is less used today, mostly Gro-Seq is used.

Q:

GRO-Seq = genomic run on sequencing

A:

With this method you can see if the increase in mRNA is due to transcriptional control.

This is very similar to the nuclear run on assay, only this one doesn't use radioactivity and uses RNA-Seq. Filtering is based on biotin-streptavidin interaction.


  • Isolate nuclei, elongate transcript in the presence of chemically modified UTP (e.g. bio-UTP)
  • Isolation of chromatin-associated, nascent mRNA using the chemical modification of U (e.g. with Streptavidin)
  • Reverse transcription -> RNA-Seq (not on total cellular RNA analysis but on nascent transcripts only! -> this corresponds to transcription rate)
  • Bioinformatic analysis of transcript frequency



Q:

Accuracy of tRNA synthetases

A:

Because of the similarity of some amino acids, finding the accurate one is a difficult task for the enzyme. Specificity thereby is controlled by proofreading reactions. After the amino acid has been linked to the AMP on the synthesis site and the tRNA binds to the enzyme, the enzyme runs a second amino acid proofreading step. It tries to force the amino acid into a second pocket, the editing site, which excludes the correct amino acid but is open for the similar ones. In the case that an incorrect amino acid is bound to the AMP or tRNA, this amino acid will be transferred to the editing site of the enzyme, where it will be removed by hydrolysis. The tRNA charging makes one mistake approximately in 40000 couplings.

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