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RNA Synthesis and Processing

Chapter Resources: Lieberman et al,2007,Marks Essential Medical Biochemistry, 2nd Edition, Lippincott Williams & Wilkins

Key points

Transcription- process by which the synthesis of RNA molecules from DNA is initiated and terminated The enzyme RNA polymerase transcribes genes into a single-stranded RNA. The RNA produced is complementary to one of the strands of DNA, which is known as the template strand. The other DNA strand is the coding, or sense strand. Bacteria contain a single RNA polymerase; eukaryotic cells utilize 3 different RNA polymerases. The DNA template is copied in the 3 to 5 direction and the RNA transcript is synthesized in the 5 to 3 direction. In contrast to DNA polymerases, RNA polymerases do not require a primer to initiate transcription, nor do they contain error-checking capabilities. Promoter regions are specific sequences in DNA that determine where on the DNA template RNA polymerase binds to initiate transcription. Transcription initiation requires a number of protein factors to allow for efficient RNA polymerase binding to the promoter. Other DNA sequences, such as promoter-proximal elements and enhancers, affect the rate of transcription initiation through the interactions of DNA-binding proteins with RNA polymerase and other initiation factors. Eukaryotic genes contain exons and introns. Exons specify the coding region of proteins, whereas introns have no coding function. The primary transcript of eukaryotic genes is modified to remove the introns (splicing) before a final, mature mRNA is produced.

Transcription in eucaryotes
Temporal and spatial separation of transcription and translation allows intricate gene expression regulation Contributes to richness and diversity of eucaryotic form and function Transcription is carried out by RNA polymerases RNA polymerases differ in templates specificity, localisation and susceptibility to inhibitors

Eucaryotic RNA polymerases


Type RNA polymerase RNA polymerase RNA polymerase RNA polymeraseV Localisation nucleus cytoplasm cytoplasm nucleus Template large rRNA genes mRNA precursors tRNA, 5S rRNA, small RNA small nuclear RNA insensitive xxx high

RNA polymerases

RNA polymerases recognize the start point for transcription of each gene and the appropriate strand of DNA to use as a template. A gene is a segment of DNA that functions as a unit to generate and regulate the expression of an RNA product or through the processes of transcription and translation, a polypeptide chain RNA polymerase is sensitive to signals that reflect the need for the gene product and control the frequency of transcription. A region of regulatory sequences called the promoter (often composed of smaller sequences called boxes or elements), usually contiguous with the transcribed region, controls the binding of RNA polymerase to DNA and identifies the start point The frequency of transcription is controlled by regulatory sequences within and near the promoter (promoter-proximal elements) and by other regulatory sequences, such as enhancers, that may be located at considerable distances, sometimes thousands of nucleotides, from the start point. Both the promoter-proximal elements and the enhancers interact with proteins which stabilize RNA polymerase binding to the promoter.

Overview

Transcription factors

Accessory DNA binding proteins which cross-talk. Binds and recognises specific sequences on the DNA. Include promoters, enhancers and silencers. They stimulate distinct sets of genes. Act like passwords that unlock multiple keys giving RNA polymerase access to specific genes.

Promoters
specific sequences in DNA that determine the start point for transcription. TATAAA box has a centre at -25 eucaryotes and -10 in bacteria Also known as TATA box Rich in AT base pairs AT base pairs in DNA are joined by only two hydrogen bonds, while GC pairs have three hydrogen bonds. Therefore, in AT-rich regions of DNA, the two strands can be separated more readily than in regions that contain GC base pairs. CAAT box variable -75 to -80 GC box variable -40

Enhancers
Regulatory sequences outside the actual promoter Binds transcription factors to increase gene expression Increase basal level of transcription and stimulate transcription Location varies suggesting they are recognised by different specific proteins Function in a forward/reverse orientation; upstream/downstream several kilobases away from the gene Other enhancers are tissue specific eg Ig enhancers function in B lymphocytes and cells with cognate proteins

RNA processing

RNA processing involves: capping, polyadenylation, splicing, editing and silencing

Capping
Capping of the primary transcript synthesized by RNA polymerase II occurs at its 5 end as it is being transcribed. The linkage formed is an unusual 5 -5 triphosphate bridge between a guanosine residue and the 5 termini of the original transcript. The now terminal guanine (part of the cap) is frequently methylated at position 7 This cap seals the 5 end of the primary transcript and decreases the rate of degradation. It also serves as a recognition site for the binding of the mature mRNA to a ribosome at the initiation of protein synthesis. Important for splicing, enhances mRNA translation rRNA, tRNA are not capped

Polyadenylation
Addition of A Poly(A) Tail Not encoded in DNA After the RNA polymerase transcribes the stop codon for protein translation, it passes a sequence called the polyadenylation signal (AAUAAA). It continues past the polyadenylation signal until it reaches an unknown and possibly unspecific termination signal many nucleotides later. the primary transcript is released from the RNA polymerase elongation complex which cleaves the primary transcript approximately 10 to 20 nucleotides downstream, thereby forming the 3 end. Following this cleavage, a poly(A) tail over 200 nucleotides in length is added to the 3 end. there is no sequence in the DNA template that corresponds to this tail; it is added post transcription. ATP serves as the precursor for the sequential addition of the adenine nucleotides. They are added one at a time, with poly(A) polymerase catalyzing each addition. The poly(A) tail is a protein binding site that protects the mRNA from degradation.

Splicing of RNA
Removal of Introns Eukaryotic pre-mRNA transcripts contain both exons and introns. Exons appear in the mature mRNA; introns are removed from the transcript and are not found in the mature mRNA. Introns therefore do not contribute to the amino acid sequence of the protein. Some genes contain 50 or more introns. These introns are carefully removed from the pre-mRNA transcript and the exons spliced together, so that the appropriate protein is produced from the gene. The consensus sequences at the intron/exon boundaries of the premRNA are AGGU (AGGT in the DNA). introns begin with a 5 GU and end with a 3 AG . Mutations in splicing sites may result in aberrant splicing

Alternate splicing pathways


Since not every combination of 5GU and 3 AG results in a functional splice site, clearly other features within the exon and/or intron help to define the appropriate splice sites. A complex structure known as a spliceosome ensures that exons are spliced together with great accuracy . Small nuclear ribonucleoproteins (snRNPs), are involved in formation of the spliceosome. Exon shuffling of troponin T: has 18 exons of which 11 are always used and the rest are shuffled to create diversity Exon shuffling may speed up evolution Trans-splicing: typical of primitive worms, involves gene shuffling Alternate tail-site selection: involves poly(A) addition signals Alternate promoter selection

Antibiotics as inhibitors
Rifamycin streptomyces inhibits initiation Rifampicin specifically binds to the -subunit of RNA polymerase Actinomycin-D streptomyces binds to DNA and prevents transcription

Epigenetics
Heritable changes in the phenotype of a cell or organism not caused by the genotype Epigenetic marks, such as the modifications of the histones, are also important for the specialisation of the body cells. Research into epigenetics has shown that environmental factors affect characteristics of organisms. These changes are sometimes passed on to the offspring They are preserved during cell division and are passed on to the daughter cells Epigenetics examines the inheritance of characteristics that are not set out in the DNA sequence. important factors are the histones, a kind of packaging material for the DNA, in order to store DNA in an ordered and space-saving way. Depending on the chemical group they carry, if they are acetylated or methylated, they permanently activate or deactivate genes. Environment affects inheritance

Implications of epigenetics
Dictates cell fate Mammalian development Aging Diseases Heritable