Gene expression 2 Flashcards
Protein subunits of RNA polymerase?
AlphaX2
Beta’
Beta
Omega
Subunits of holo RNA polymerase?
AlphaX2
Beta’
Beta
Omega
Sigma
In prokaryotic transcription, what enzyme recognises promoter?
Holo RNA polymerase.
What binds the consensus sequence in the core promoter?
Sigma factor.
Consensus element?
-35 and -10 elements
What is found to be the optimum spacing in consensus?
17
Open complex formation?
Promoter opening allows for exposure of template strand for complementary RNA strand.
This formation is in equilibrium.
Anchored transcription?
Only short transcripts can be synthesized whilst sigma factor remains bound to the promoter.
Promoter escape?
Sigma factor released from the promoter.
Elongation factor binds and transcription proceeds.
Enzyme becomes processive and makes longer transcripts.
Transcription termination in prokaryotic cells?
The palindromic sequence in the RNA forms a hairpin loop causing RNA polymerase to stall.
The u-rich stretch downstream of the termination signal allows the transcript to fall off the template, thereby terminating.
4 rough steps of prokaryotic transcription.
Open complex.
Anchored transcription.
Promoter escape.
Termination.
Rho-independent termination?
A terminating hairpin forms on the mRNA interacting with the NusA protein to stimulate release of transcript from the RNA polymerase complex.
Rho-dependent termination?
The Rho protein binds at the upstream rut site, translocates down the mRNA, and interacts with the RNA polymerase complex to stimulate release of transcript.
RNA polymerase I structure?
5.8S, 18S and 28S rRNA genes.
RNA polymerase II structure?
All protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, IncRNA genes and most snRNA genes.
RNA polymerase III structure?
tRNA genes, 55rRNA genes, some snRNA genes and genes from other small RNAs.
How many subunits make up bacteria?
5
How many subunits make up eukaryotes?
12-17
What are the two important DNA sequences in Eukaryotic transcription?
Core promoters
Promoter proximal and distal elements.
Core promoters role in eukaryotic transcription?
Recognised by general transcription factors that recruit RNA polymerase.
Steps of general transcription at core promoters?
-RNA polymerase itself cannot find promoters.
-TFIID makes multiple specific contacts with core promoter elements such as TBP interacting with TATA box.
-TFIID recruits TFIIB.
TFIID is critical for recruitments of RNA polymerase II (with TFIIF)
-This complex cannot initiate transcription even though RNA polymerase II is there.
-Once TFIIE and TIFFIH have joined a transcription initiation component pre-initiation complex has formed.
-TFIIE and TFIIH stimulate and stabilize promoter opening to allow initiation of transcription by RNA polymerase.
Role of promoter proximal and distal elements in eukaryotic transcription?
Regulatory sequences and binding sites for transcriptional activators and repressors.
Order of TF complex in general transcription at core promoter?
TFIID
TFIIB
TFIIE TIFFIH
Role of TFIIB
Recruits RNA polymerase.
Role of TFIID?
Makes multiple specific contacts with core promoter elements.
Recruits TFIIB
What happens when TFIIE and TIFFIH joins.
Transcription initiation competent pre-initiation complex forms.
Stimulates and stabilizes promoter opening to allow initiation of transcription by RNA polymerase.
What can affect the rate of transcription initiation?
-Sequences that bind specific transcription factors may be far from actual transcription site
-DNA bending allows specific transcription factors to interact with the RNA polymerase complex and rate of transcription.
DNA binding of transcription factors?
-Specific hydrogen bond between an amino acid and a base
-A typical protein-DNA interaction might involved about 29 similar contacts.
4 dimerization domains?
-Helix-turn-helix motif
-Zinc finger motif
Leucine zippers motif
Helix-loop-helix motif
Role of dimers?
Dimers of transcription factors binds sequence-specifically to those short sequence-specifically to those short sequence elements, which are often arranged in palindrome.
What determines differential gene expression?
Transcription factors.
How in mRNA processed in eukaryotes?
-A 5’ cap (modified GTP) is added at the 5’ end, which facilitates binding to a ribosome and protects mRNA from being digested and protects mRNA from being digested by ribonucleases.
-A poly A tail is added at 3’ end: sequence AAUAAA after the last codon signals an enzyme to cut the pre-mRNA; the another enzyme added 100-300 adenine (to form tail). The tail assists in export from the nucleus and is important for stability in mRNA.
Explain how genes are split in eukaryotes?
In eukaryotes, the coding sequences are interrupted with non-coding sequences (introns) that are spliced out of the mature mRNA transcript.
Explain RNA processing?
RNA splicing removes intron sequences from newly transcribed pre-mRNAs.
The precursor mRNA transcript introns are removed by a process called RNA splicing, leaving the protein coding sequences (exons) in the mature mRNA.
Steps of RNA splicing involving the spliceosome?
- Small nuclear ribonucleoprotein particle bind to consensus sequences in RNA near 5’ donor and 3’ acceptor splice sites.
- Binding of snRNAs recruits other proteins
- A cut is made between the 5’ exon and the intron
- After the first cut at the 5’ end, the intron forms a closed loop.
- The free 3’ end OH group ,at end of cut, exons’ reacts with 5’ phosphate group of other exon.
- The 3’ exon is cleaved and spliced to the 5’ exon and the mature mRNA is exported to cytoplasm for translation
- The excised intron is degraded in the nucleus.
Explain alternative RNA splicing?
Alternative splicing can give more than one product from single genes.
Alternative splicing can be regulated to give differential gene expression.
Eukaryotic and prokaryotic order of transcription and translation.
Prokaryotic cells are translated at the same time and transcribed.
Eukaryotic cells RNA transcripts must be processed and transported to cytoplasm before being translated. Leaves nucleus through nuclear pore complexes.
