Transcription

Describe three ways that the RNA transcript is modified prior to translation in eukaryotes?

A 5' cap and a 3' poly-A tail are added to the mRNA. The pre-mRNA is spliced to remove the introns. This creates the mature mRNA that is ready for translation.

RNA Polymerase I and II and III

Bacteria have one type of RNA polymerase while eukaryotes have three RNA polymerase I transcribes genes that code for the large RNA molecules, called ribosomal RNA (rRNA), that are found in ribosomes. RNA polymerase II transcribes protein-coding genes and creates messenger RNA (mRNA). RNA polymerase III transcribes genes that code for transfer RNAs (tRNAs) that play a key role during translation.

During elongation (transcription)

During elongation, RNA polymerase moves along the DNA template 3′ to 5′ and adds new nucleotides to the 3′ end of the RNA transcript (Figure 3). Nucleotides are added to the RNA by complementary base pairing to the DNA template strand. The base pairing during transcription is the same as in DNA base pairing, except that RNA contains uracil instead of thymine. RNA polymerase catalyzes the formation of phosphodiester bonds (make up backbone of DNA) between these monomers as the transcript is created.

transciption initiation

During initiation, with the help of additional factors, RNA polymerase binds with the DNA and the DNA unwinds. During the elongation phase, RNA polymerase moves along the DNA template and creates the RNA transcript. Finally, termination occurs when RNA polymerase reaches the termination site and the RNA transcript is released. The initiation of transcription requires a special DNA sequence called a promoter. The promoter tells the RNA polymerase where to start transcription and this site is called the transcription initiation site or transcription +1 site. The promoter also tells RNA polymerase which DNA strand to use as the template. The sequences and factors involved in initiation differ between prokaryotic and eukaryotic transcription.

codon strand

During transcription, only one strand of the DNA is used as a template to create the RNA molecule. This is called the template strand. The other strand is called the non-template or coding strand. It is called the coding strand because its sequence will match the sequence of the newly created RNA strand. But the coding strand is not an exact match to the RNA because RNA uses the nucleotide uracil (U) in place of thymine (T).

Transcription initiation differs in prokaryotes and eukaryotes

In addition to RNA polymerase, there are other factors that are required for transcription. (transcription factors) In prokaryotes, a protein subunit called sigma binds to the core RNA polymerase to create what is known as a holoenzyme. It is the sigma portion of the holoenzyme that binds to the promoter to initiate transcription. There are a variety of sigma proteins, each with a slightly different structure. By pairing with different sigma proteins, RNA polymerase may bind to different promoters. The genes transcribed by the holoenzyme are dependent on which sigma protein is present in the holoenzyme. Eukaryotes also require additional factors for RNA polymerase to bind to the DNA. These proteins are called basal transcription factors. These proteins assemble at the promoter first, and then RNA polymerase binds to form what is known as the transcription initiation complex. Once the holoenzyme (in prokaryotes) or transcription initiation complex (in eukaryotes) is bound to the promoter, the DNA helix unwinds

pre mRNA to mature mRNA

In bacteria, the RNA is ready for translation as soon as it is transcribed. However, in eukaryotes the mRNA must undergo processing before translation may begin. (needsto be mature before it leaves nucleus to cytoplasm). Before this processing occurs, the mRNA transcript is known as pre-mRNA. a modified guanine (G) nucleotide is added to the 5′ end of the transcript, creating the 5′ cap. The 5′ cap helps the transcript bind to the ribosome for translation. It also helps protect the mRNA from degradation by ribonucleases. A poly-A tail is added to the 3′ end of the pre-mRNA transcript. The poly-A tail is made up of 50-300 adenine (A) nucleotides. The poly-A tail aids in the export of the mRNA to the cytoplasm for translation. And like the 5′ cap, the poly-A tail protects the mRNA from degradation.

exon and introns

In eukaryotes, before translation can occur, introns must be removed and the exons combined to form mature mRNA. Large portions of the pre-mRNA molecule are removed before the mRNA is exported from the nucleus. The segments of the pre-mRNA that are included in the final mRNA molecule are called exons. The non-coding segments that are removed are called introns. A spliceosome is created at each exon-intron junction. The spliceosome cuts the pre-mRNA, removes the intron and joins the exons together. In this way, all of the introns are removed and the exons spliced together to form the mature mRNA that is ready for translation.

alternative RNA splicing

One effect of RNA splicing is the ability to change which sequences are treated as exons, and therefore create different mature mRNA molecules from the same gene. This is known as alternative RNA splicing. Proteins are made up of structural and functional regions called domains. For example, one domain may contain an active site while another may contain a binding site. In many cases, different domains are coded for by different exons. By using alternative splicing, the same gene is able to produce a variety of proteins that contain different domains

Transcription Termination

Termination occurs once RNA polymerase reaches a specific sequence on the DNA template. In bacteria, one type of terminator sequence codes for a stretch of RNA that, once transcribed, creates a hairpin by folding back on itself. The short hairpin is created by base pairing of complementary bases in the RNA, and this structure combined with weak base pairing of downstream Us on the RNA and As on the DNA disrupts the association between the RNA polymerase and the RNA transcript. In eukaryotes, termination occurs once a sequence called a polyadenylation signal (AAUAAA) is transcribed. Once RNA polymerase reaches 10-35 base pairs downstream from the polyadenylation signal, the transcript is released from RNA polymerase.

transcription initiation complex

The combination of transcription factors and RNA polymerase that assembles at the promoter of a gene prior to transcription initiation.

Transcription versus DNA Replication

The process of transcription creates an RNA version of the information coded in the DNA. Transcription is similar to DNA replication in that the DNA is unwound and a polymerase reaction adds the appropriate nucleotide substrates to the growing nucleotide chain. However, there are several key differences between DNA replication and transcription. During transcription, only one strand of the DNA is used as a template to create the RNA molecule. RNA polymerase does not need a primer to start transcription. The stretch of DNA that is transcribed into RNA is known as the transcription unit.

Transcription has three distinct phases:

initiation, elongation and termination.

What is the function of the sigma factor in transcription?

sigma factors enable specific binding of RNA polymerase to promoters.