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TRANSCRIPTION IN EUKARYOTES Transcription is the synthesis of RNA from a DNA template and is catalyzed by enzymes known as RNA polymerase.

This process occurs in the nucleus of the eukaryotic cells. During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. RNA polymerase opens up the double strand so that the DNA bases can be accessed to direct the order of bases on the RNA. There are 3 types of RNA polymerase; RNA polymerase 1 responsible for synthesis of rRNA RNA polymerase 11 responsible for synthesis of mRNA RNA polymerase 111 responsible for synthesis of tRNA The eukaryotic RNA polymerase is larger than that of Prokaryotes but their subunits have similar roles. The process of transcription has 3 recognizable steps; initiation, elongation and termination. Initiation This involves recognition of the start point of transcription usually located upstream of the gene of interest. This is determined by a special sequence known as a promoter sequence which is recognized by the RNA polymerase. A Promoter sequence is a unidirectional sequence found on one strand of the DNA that instructs the RNA polymerase in both where to start synthesis and in which direction synthesis should continue. In eukaryotes it is found at -30, -75 and -90 base pairs upstream from the start site of transcription. In addition to promoters, eukaryotic genes also have regulatory regions called enhancers. Both elements (promoter and enhancer) are required for correct expression of eukaryotic genes. As a result of this added complexity, eukaryotic RNA polymerases do not have anything equivalent to the sigma subunit found in prokaryotic RNA polymerases. Instead, eukaryotes have groups of transcription factors, which are proteins, independent of the RNA polymerases that recognize promoter and enhancer sequences. The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found -30 base pairs from the start site of transcription. The TATA box is the binding site for a transcription factor known as TATA binding protein (TBP), which is itself a subunit of another transcription factor called Transcription Factor II D (TFIID). TFIID and the other factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and TFIIJ) form a complex on the DNA that recruits RNA polymerase II to the promoter, and promotes initiation of transcription. These

transcription factors are sufficient to get a basal (minimal) level of transcription. Other transcription factors binding to other promoter and enhancer elements are necessary for higher levels of transcription. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex known as an Open Promoter Complex. Enlongation One strand of DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. Most RNA molecules start with a G or A and unlike DNA synthesis a primer is not required. As transcription proceeds, RNA polymerase traverses the template strand opening the helix and uses base pairing complimentarity with the DNA template to create an RNA copying in the 3- 5 direction. Only a small scale of DNA is unwound at a time to protect DNA from undesired cleavage. Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene. Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure. Termination Eukaryotic genes have no strong termination sequences like prokaryotes. Instead, RNA polymerase II continues transcribing up to 1000 to 2000 nucleotides beyond where the 3' end of the mature mRNA will be. The actual 3' end will be determined during RNA processing.

GENE PROCESSING OR EDITING Eukaryotic class II transcripts are processed in order to produce the final mRNA. Processing of the initial transcript includes capping, polyadenylation and intron removal. Ribosomal and transfer RNAs are also processed, but differently; they are neither capped nor poly adenylated. Capping of the RNA

This occurs at the 5' end and a methylated guanine nucleotide is added to the transcript in a 5' to 5' phosphodiester linkage (it's like a nucleotide added to the 5' end in the backwards direction). This 'cap' is important for recognition of the mRNA by ribosomes during translation. Polyadenylation

This involves cleavage of the RNA to produce the proper 3' end, and addition of a string of adenine nucleotides. The position of the 3' end is determined by a sequence within the RNA itself. This sequence, AAUAAA, is known as the polyadenylation signal. When this signal is recognized by the appropriate enzymes, the RNA is cleaved 10 to 30 nucleotides downstream of the signal, and a series of adenine nucleotides is added. Polyadenylation is done without a template, the As are simply added one after another to the 3' end of the RNA. This poly (A) tail, which averages about 200 nucleotides in length, helps protect the RNA from degradation, and plays other regulatory roles. Intron removal

Introns in some RNAs (particularly mitochondrial RNAs) are capable of self-splicing (or autocatalytic splicing). In these splicing reactions, no protein enzyme is required - the enzyme activity resides within the intron RNA itself! Such RNA enzymes are termed ribozymes. Class II RNAs (pre-mRNAs) from most eukaryotes, however, do require protein enzymes to remove their introns. The splicing of these RNAs is carried out by large protein/RNA complex called spliceosomes. Spliceosomes are made up of five different snRNPs called U1, U2, U4, U5, and U6. Each snRNP consists of a specific small nuclear RNA (snRNA) molecule complexed with protein. Spliceosomes are able to detect intron/exon boundaries, cleave the RNA at the appropriate point, and join adjacent exons together to produce the mature mRNA. Differences between transcription in plants and animals Vertebrate genes can be quite large, often spanning tens of thousands of base pairs and usually separated by numerous large introns, whereas plant genes tend to be much smaller (between 12 kilobases) with fewer and smaller introns. Second, plant transcripts retain introns more often than do animal transcripts (30% of all genes in the model plant, Arabidopsis, compared to 10% in humans).

Differences between transcription in Prokaryotes and Eukaryotes In prokaryotes, transcription occurs in the cytoplasm since they dont have a well defined nucleus while in eukaryotes it occurs in the cell's nucleus. Prokaryotic DNA is much more accessible to RNA polymerase than DNA in eukaryotes. Eukaryotic DNA is wrapped around proteins called histones to form structures called nucleosomes. While RNA polymerase interacts directly with prokaryotic DNA, other proteins mediate the interaction between RNA polymerase and DNA in eukaryotes. Prokaryotic RNA polymerase is of one type and made up of 5 polypeptide subunit; 2, and 1 are the catalytic subunits while is the regulatory subunit. It is known as a core enzyme if the sigma unit is missing and a holo enzyme if the sigma unit is present. The sigma subunit is the one that recognizes the promoter sequences -35 and -10 upstream of the gene of interest. Promoter specific initiation in eukaryotes requires more than a dozen basal initiation factors while in prokaryotes it is only a single polypeptide; the sigma subunit. Eukaryotic mRNA contain no ShineDalgarno sequence to show the ribosomes where to start translating instead most eukaryotic mRNA have caps at their 5` which directs initiation factors to bind and begin searching for an initiation codon. Eukaryotic protein synthesis initiation begins with methionine not N formyl methionine Termination of transcription involves formation of a hair pin loop structure and can be dependent or independent of the rho protein factor. Rho-independent terminators have a characteristic structure, which features; a strong G-C rich stem, a hair pin loop and a sequence of 46 U residues in the RNA which are transcribed from a corresponding stretch of Adenines in the template. Rho-factor-dependent terminators are less well defined. The mRNA produced as a result of transcription is not modified in prokaryotic cells.