分子生物学-05真核转录

1、1. RNA polymerase: 3 types RNA polymerase I: in nucleolus; rRNA transcription; the most cellular RNA synthesis. Did not inhibited by a-amanitinRNA polymerase II: in nucleoplasm , heterogeneous nuclear RNA (hnRNA) transcription (the precursor for mRNA). inhibited by low concentrations of a-amanitin.

2、RNA polymerase III: a minor enzyme activity. in nucleoplasm; tRNAs and other small RNAs (5S rRNA)transcription. inhibited or not by a-amanitin. All eukaryotic RNA polymerases are large proteins, 500 kD, 8-14 subunits. RNA polymerases IIThe largest subunit has a carboxy-terminal domain (CTD), which c

3、onsists of multiple repeats of a consensus sequence of 7 amino acids. The sequence is unique to RNA polymerase II. The RNA polymerase activities of mitochondria and chloroplasts are smaller, and resemble bacterial RNA polymerase. 2. Promoter RNA polymerase I promoter has two sequence componentsCore

4、promoter: 70 bp.UBF1: a single polypeptide, it binds to both regions (GC-rich), after which SL1 can bind. RNA pol I then binds to the core promoter. UBF1: SL1: 4 subunits, one subunit-TBP interacts with RNA polymerase. SL1 resembles sigma factor.Deletion analysis shows that the promoter for 5S RNA g

5、enes is internal; initiation occurs a fixed distance (55 bp) upstream of the promoter. RNA polymerase III has a internal promoter RNA polymerase III has a internal promoter Promoters for RNA polymerase III consist of bipartite sequences: boxA separated from either boxC or boxB. Or they may consist o

6、f separated sequences upstream of the startpoint (Oct, PSE, TATA). Promoter structure of RNA polymerase III The binding of the assembly factors TFIIIA and TFIIIC, the initiation factor TFIIIB, and RNA polymerase III. Promoter structure of RNA polymerase III TFIIIA: a zinc finger. TFIIIB: three subun

7、its.TFIIIC: at least 5 subunits, 500 kD.TFIIIA and TFIIIC are assembly factors, whose role is to assist the binding of TFIIIB at the right location. TFIIIB functions as a positioning factor, responsible for localizing RNA polymerase correctly. Like SL1 at the pol I promoter, it resembles a sigma fac

8、tor. Promoter structure of RNA polymerase II One upstream promoter, with relatively fixed location.TATA box: 25 bp upstream, 8 bp consensus sequence consists entirely of AT. The TATA box tends to be surrounded by GC-rich sequences. It is almost identical with the -10 sequence found in bacterial prom

9、oters. The minority of promoters that do not contain a TATA element are called TATA-less promoters. TBP: TATA-binding protein, 30 kD.TAFs: for TBP-associated factors. The assembles at the promoter Promoters for RNA polymerase II have short sequence elements CAAT box : -75, CAAT.a common elements, fu

10、nctions in either orientation. To increases promoter strength. GC box:-90, GGGCGG. Often multiple copies. in either orientation. a relatively common promoter component. Promoters for RNA polymerase II have short sequence elements Promoters for RNA polymerase II have short sequence elements 3. Enhanc

11、er bidirectional elements that assist initiation function in either orientation and in any location (upstream or downstream) relative to the promoter An enhancer in the virus SV40 It contains two identical sequences of 72 bp each, repeated in tandem 200 bp upstream of the startpoint of a transcripti

12、on unit. Each 72 bp repeat contains a copy of the enhancer 4. RNA processing mRNA processing 5 Capping 3 Cleavage and polyadenylation Splicing Pre-mRNA methylation mRNA editing Splicing Splicing The ends of nuclear introns are defined by the GT-AG rule. (GT-AG rule). Splicing occurs in two stages, i

13、n which the 5 exon is separated and then is joined to the 3 exon. 5 Capping cap 0: in all eukaryotes; guanine-7-methyltransferase. cap 1: the predominant type of cap in all eukaryotes except unicellular organisms; 2-O-methyl-transferase. cap 2: 10-15% of the total capped population. Poly(A) tial add

14、s 200 A residues to the free 3-OH end of the mRNA; catalyzed by the enzyme poly(A) polymerase the poly(A)-binding proteins (PABP) binds on poly(A) tail, One PABP monomer of 70 kD is bound every 10-20 bases of the poly(A) tail. Removal of the poly(A) tail precedes the degradation of certain mRNAs; Th

15、e ability of the poly(A) to protect mRNA against degradation requires binding of the PABP. Removal of poly(A) inhibits the initiation of translation. However, there are many examples in early embryonic development where polyadenylation of a particular mRNA is correlated with its translation. snRNA (

16、small nuclear RNA) is any one of many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions. (100-300 bases and 105-106 molecules per cell. In natural state, they exist as ribonucleoprotein particles (snRNP).The spliceosome contai

17、ns snRNA Spliceosome: proteins and RNAs existed as ribonucleoprotein particles. The snRNPs involved in splicing are U1, U2, U5, U4, and U6. Each snRNP contains a single snRNA and several (20) proteins. The U4 and U6 snRNPs are usually found as a single (U4/U6) particle. The snRNPs together contain 40 individual proteins. Alternative splicing involves differential use of splice junctions Alternative splicing involves differential use of splice junctions cis-splicing and trans-splicing reactions Splicing usua