分子生物学6真核生物的转录及其调控

2. RNA Pol I promoters in human cells are best characterized.
•Core element: -45 to +20, sufficient for transcription initiatiation.
•UCE: -180 to -107, to increase the transcription efficiency.
SL1 consists of 4 proteins
1. TBP (TATA-binding protein)
a factor also required for initiation by RNA Pol II and III. A critical general factor in eukaryotic transcription that ensures RNA Pol to be properly localized at the startpoint.
RNA Pol I genes:
the ribosomal repeats
Ribosomal RNA Genes & nucleolus
1. A copy of 18S, 5.8S and 28S rRNA genes is organized as a single transcription unit in eukayotes. A 45S rRNA transcript (~13 000 nt long) is produced during transcription, which is then processed into 18S, 5.8S and 28S rRNA.
RNA Pol I promoters
1. Generally consists of a bipartite sequence in the region preceding the start site, including core element and the upstream control elements (UCE).
RNA Pol I Nucleoli Most rRNAs gene Insensitive
RNA Pol II
Nucleoplasm All protein-coding genes and some snRNA genes
Very sensitive
RNA Pol III Nucleoplasm tRNAs, 5S rRNA, U6 snRNA and other small RNAs
5. The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter (In vitro results).
•Both regions are rich in G:C, with ~85% identity.
Upstream binding factor (UBF)
A specific DNA-binding protein that binds to UCE, as well as a different site in the upstream of the core element, causing the DNA to loop between the two sites. (two binding sites have no obvious similarity) •UBF is essential for high level of transcription, and low level of expression occurs in its absence.
Simple initiation
TIF -1(homolog of SL-1) binds to the promoter RNA Pol
I bind
TIF-1 remains bound and the RNA Pol I is
released for elongation.
RNA Pol III genes: 5S and tRNA transcription
4.The arrays of rRNA genes (rRNA cluster) loop together to form the nucleolus and are known as nucleolar organizer regions.
5. During active rRNA synthesis, the pre-rRNA transcripts are packaged along the rRNA genes, visualizing in the electronic microscope as “Christmas tree structures”.
2. Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.
3. Continuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes.
Moderately sensitive
α-amanitin
The sensitivities of three kinds of RNA polymerases on αamanitin
RNA polymerase subunits
Each eukaryotic polymerase contains 12 or more subunits. –the two largest subunits are similar to each other and to the β’and β subunits of E. coli RNA Pol. –There is one other subunit in all three RNA Pol homologous to α subunit of E. coli RNA Pol. –Five additional subunits are common to all three polymerases. –Each RNA Pol contains additional four or seven specific subunit.
3. Three eukaryotic polymerases transcribe different sets of genes. The activities of these polymerases are distinguished by their sensitivities to the fungal toxin α-amanitin.
Selectivity factor 1 (SL1)
1. Does not bind to promoters by itself 2. Binds to and stabilizes the UBF-DNA complex. 3. Interacts with the free downstream part of the core element. 4. Recruit RNA Pol I to bind and to initiate the transcription.
4. In addition, eukaryotic cells contain additional RNA Pols in mitochondria and chloroplasts.
Three eukaryotic polymerases
Typeቤተ መጻሕፍቲ ባይዱ
Location Substrate
α-amanitin
Tyr-Ser-Pro-Thr-Ser-Pro-Ser 3. This repeated sequence is known as carboxyl terminal domain (CTD) 4. The CTD sequence may be phosphorylated at the serines and some tyrosines
Transcription in Eukaryotes
The three RNA Polymerases
characterization and function
Main Features of eukaryotic transcription
1.The mechanism of eukaryotic transcription is similar to that in prokaryotes. 2.A lot more proteins are associated with the eukaryotic transcription machinery, which results in the much more complicated transcription.
RNA polymerase activities
1. Transcription mechanism is similar to that of E. coli polymerase. Direction: 5’ 3’ 2.Different from bacterial polymerasae, they require accessory factors for DNA bind