TRANSCRIPTION IN EUKARYOTES

Name : Shilpi Roll No: 1491 B.Sc. Life Sc. 2nd year , 4th sem Molecular Biology

INTORODUCTION TO TRANSCRIPTION

Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement.

EUKARYOTIC

Eukaryotic transcription is more complex than prokaryotic transcription. For instance, in eukaryotes the genetic material (DNA), and therefore transcription, is primarily localized to the nucleus, where it is separated from the cytoplasm (in which translation occurs) by the nuclear membrane. DNA is also present in mitochondria in the cytoplasm and mitochondria utilize a specialized RNA polymerase for transcription. This allows for the temporal regulation of gene expression through the sequestration of the RNA in the nucleus, and allows for selective transport of RNAs to the cytoplasm, where the ribosomes reside.

STEP IN TRANSCRIPTION

Transcription is explained easily in 4 or 5 steps, each moving like a wave along the DNA. RNA polymerase moves the transcription bubble, a stretch of unpaired nucleotides, by breaking the hydrogen bonds between complementary nucleotides. RNA polymerase adds matching RNA nucleotides that are paired with complementary DNA bases. RNA sugar-phosphate backbone forms with assistance from RNA polymerase. Hydrogen bonds of the untwisted RNA+DNA helix break, freeing the newly synthesized RNA strand. If the cell has a nucleus, the RNA is further processed (addition of a 3' poly-A tail and a 5' cap) and exits through to the cytoplasm through the nuclear pore complex.

A.
Carried out by three different RNA polymerases, each of which contains more than10 subunits (i.e., these are very complex enzymes). All three require several assisting proteins, collectively called transcription factors. RNAP I all ribosomal RNAs (5.8S, 18S, 28S) except for 5S found in the nucleolus RNAP II all nuclear genes encoding proteins (mRNAs) found in the nucleus RNAP III all A, the 5S rRNA, and all snRNAs found in the nucleus

B.
1.

Initiation of RNA synthesis: For RNAP II (protein-coding genes), initiation requires several transcription factors that assist binding to promoter sites. Promoters sites recognized by RNAP II (and associated protein factors) are several conserved elements that are located upstream from the transcription start point (the +1 base). The consensus sequences of the conserved elements are
a. b. c.

30 = TATAAAA 80 = GGCCAATCT GGGCGG [GC box] & positions and in ATTTGCAT [octamer box]

[TATA homology or Goldberg-Hogness box] [CAAT box] often present but occur in different different copy numbers

2.

3.

The TATA homology is found in all eukaryotic promoters known to date. The remaining consensus sites are found but not necessarily in the same promoter. All consensus sites affect binding efficiency of RNA polymerase/transcription factors. RNAP I and RNAP III utilize some of the same transcription factors as RNAP II but the promoters are quite different. RNAP III utilizes internal promoters (i.e., within the transcriptional units).

C.
Elongation of RNA via RNAP II (protein-coding genes): Elongation of the RNA chain is similar to that in prokaryotes except that a 7-methyl guanosine (7MG) cap is added to the 5 end when the growing RNA chain is fairly short (20-30 bases in length).
A.

The 7-MG cap is attached by an unusual 5-5 triphosphate linkage and serves to protect the growing RNA from degradation by nucleases. This capping is part of RNA processing in eukaryotes.

D.
Termination of RNA synthesis: 1. Transcription by RNAP II (for protein-coding genes) is not really terminated, in the sense that transcription continues for 1,000 - 2,000 bases after or downstream from the site that ultimately will become the 3 end of the mature transcript.
A. B. C.

The functional transcript actually results from endonucleolytic cleavage of the primary transcript. Cleavage occurs 10-30 bases downstream from the conserved sequence AAUAAA. After cleavage, an enzyme [poly(A) polymerase] adds about 200 adenine (A) bases to the 3ends. This is called polyadenylation or the addition of poly-A tails.

The function of poly-A tails is to increase stability of the transcript and to assist in transport of the mRNA from the nucleus to the cytoplasm. This is another part of RNA processing is eukaryotes.

2.

Termination of transcription via RNAPI and RNAP III is via response to discrete termination signals.

E.
Transcription enhancers and silencers: 1. These are sequences that exist upstream or downstream of the transcribe sequence, and that enhance or depress rates of transcription. 2. Most enhancers and silencers are not well characterized, can exist close to or distant (>1,000 bp) from the gene(s) they affect.

TRANSCRIPTION REGULATION

The regulation of gene expression is achieved through the interaction of several levels of control including the regulation of transcription initiation. Most (not all) eukaryotes possess robust methods of regulating transcription initiation on a geneby-gene basis. The transcription of a gene can be regulated by cis-acting elements within the regulatory regions of the DNA, and trans-acting factors that include transcription factors and the basal transcription complex.