Transcription: Transcription Products

Introduction

RNA Polymerases and the Transcription Event

Transcription Products

Introns, Exons, and Splicing hn RNA

Alternate Splicing of Exons

Course Topics

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Transcription Products

Polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs. Polycistronic mRNAs consist of a leader sequence which precedes the first gene. The gene is followed by an intercistronic region and then another gene. A trailer sequence follows the last gene in the mRNA. Examples of a polycistronic transcripts are found in the chloroplast. One region that exhibits a group of different polycistronic messages from the same region is the psbb/psbH/petB/petD region. The following table lists the genes, their products and the complex of which the product is a part.

Gene Product Complex

psbB 51 kd chl a binding protein of PSII PSII

psbH 10 kd phosphoprotein of PSII PSII

petB cytochrome b6 Cytochrome

petD subunit 4 of cytochrome b6/f Cytochrome

Although the transcripts are co-transcribed, the ratio of the two complex varies in the light and the dark as well as between the mesophyll and the bundle sheath cells. Thus some sort of regulation must exist. At least 15 different mRNAs are produced from this gene cluster.

Monocistronic mRNA is a mRNA that encodes only one protein and all eukaryotic mRNAs are monocistronic. The development of the mature monocistronic eukaryotic transcript involves several different processing steps. These steps are:

5' capping
3' polyadenylation
splicing together of exons if introns are present

Each of these steps are post-transcriptional modification steps. Thus the original transcript is not the same as the final product. All of the post-transcriptional steps occur in the nucleus of the cell and the resultant product, the mRNA, is transported to the cytoplasm for translation. So what is the original product of transcription? This product is called hnRNA. This stands for heterogeneous nuclear RNA. Not all of the hnRNA becomes a mRNA product. One question that is yet unresolved is how control is exerted over gene expression? Can control be at the steps which mature the hnRNA? How is transcription of tissue specific genes regulated? These are important question that are currently being researched.

hnRNA transcription is only a small portion of the transcription which occurs in the nucleus. Up to 90% of the transcription is for the production of rRNA. But if we limit our discussion to hnRNA some important observations can be made. First, the complexity of the hnRNA is 4 to 10 times greater than that of the mRNA. This suggest two possibilities. One, we already know and that is that splicing of genes reduces the size of the final mRNA product in comparison to the original transcript. But this does not account completely for the differences in complexity. The majority of the difference appears to be accounted for by destruction of hnRNAs before they develop into mature mRNAs. hnRNAs are a broad class of products:

Average size - 8000 - 10,000 bases
Range - 2000 - 14,000 bases

Since the average mRNA size is 1800-2000 bases, a considerable amount of trimming must occur.

hnRNA does not actually exist in the nucleus as naked RNA but is actually associated with protein. This complex is designated hnRNP and is called heteronuclear ribonuclearprotein. hnRNP is associated with the nuclear matrix. Since chromatin is attached to the nuclear matrix it seems logical that the primary transcript will also become attached to the matrix just after transcription.

The lifetime of hnRNA is quite short usually from one minute to one hour at the most. So what occurs? The first step in processing is the addition of a Cap. The cap is a 5' methyl guanosine that is added immediately after the start of transcription. Capping occurs so quickly that we rarely see the original 5' base of the message. The linkage between the 5' methyl guanosine is not the typical 5'-3' linkage but is a 5'-5' linkage. The reaction is catalyzed by the enzyme guanylyl transferase. The guanosine that is added is always methylated at the 7 position of the guanine base (7mG). This is called cap 0. In addition a methyl group is added to 2'-OH of the original base in the mRNA. This is catalyzed by 2'-O-methyl-transferase, and this methyl group is referred to as cap 1. Other methylations can occur, but we won't consider them.

About 25% of hnRNA eventually matures in polyadenylated mRNA. Not all mRNAs are polyadenylated. The histone mRNAs are a notable exception to the rule. If an inhibitor of polyadenylation is added to a reaction hnRNA is not converted into mRNA. Thus polyadenlyation is a requirement for mRNA to appear.

Termination of transcription is not understood at all. One sequence that is invariant in eukaryotic mRNA is the sequence 5'-AAUAAA-3' that is found about 10-30 bp upstream of the poly A tail. The question that this raises is whether this sequence is required for polyadenylation. Deletions or mutations of this sequence will eliminate polyadenylation. But surprising, point mutation greatly reduce the number of molecules that are cleaved. But those that are cleaved are polyadenylation. Thus, this sequence seems to be required for cleavage of the primary transcript. The poly-A tail is added by the enzyme poly(A) polymerase.