TRANSCRIPTION AND PROCESSING OF RNA 1. The steps of gene expression. 2. General characterization of transcription: steps, components of transcription apparatus. 3. Transcription of eukaryotic structural genes. 4. Processing of eukaryotic mrnas. 5. Particularities of transcription of trna and rrna genes in eukaryotes. 6. Transcription of mitochondrial genome. 7. Particularities of transcription in prokaryotes. All of information about organisms is stored in DNA. Realization of information is made by gene expression. Gene expression represents the conversion of genetic information encoded in a gene into RNA and protein, by transcription of a gene into RNA and (in the case of protein-coding genes) the subsequent translation of mrna to produce a protein. In both eukaryotes and prokaryotes there are some steps of gene expression. DNA replication DNA repair Genetic recombination Codons DNA transcription RNA synthesis Protein synthesis DNA RNA Protein The steps of gene expression in prokaryotic cells: - Activation and transcription of genes - Translation of mrna synthesis of proteins - Conformation post-translational modification of proteins. The steps of gene expression in eukaryotic cells: - Activation and transcription of genes - Processing of RNAs - Export of RNAs from nucleus to cytoplasm - Translation of mrna synthesis of proteins - Conformation post-translational modification of proteins. Amino acids Transcription is the first step in gene expression. Transcription represents the process of complimentary synthesis of RNA from a DNA template. Stretch of DNA that is transcribed as a single continuous RNA strand, a transcript, is called a transcription unit. A unit of transcription may contain one or more sequences encoding polypeptides (translational open reading frames (ORF) or cistrons). In prokaryotes polycistronic mrnas are common. In eukaryotes, monocistronic mrnas are the general rule, but some transcription units encode more than one polypeptide as a consequence of alternative transcriptional start sites and/or alternative pathways of RNA splicing or other types of post- transcriptional RNA processing. Components required for transcription: - DNA molecule containing regulatory (promoter, terminator) and coding sequences - RNA-polymerazes - Specific transcription factors - General transcription factors - NTP (ATP, GTP, CTP, UTP). Transcription is the principal point at which gene expression is controlled in both prokaryotes and eukaryotes. There are three steps in the process of transcription:
- Initiation (the most important) - Elongation - Termination Transcription and processing RNA-polymerases RNA-polymerases are enzymes which catalyze the synthesis of RNA using DNA as template. Direction of synthesis of RNA is 5' 3' and DNA is reading in direction 3' 5'. The strand of DNA which serves as template is named sense chain, and the other strand, identical with RNA, is named coding strand (Fig. 1). Fig. 1. Transcription of RNA The prokaryotic cells have only one type of RNA-polymerase, which synthesis all kinds of RNAs. The eukaryotic cells have three distinct classes of RNA-polymerases: - RNA-polymerase I the most active polymerase (50-70% of all cellular RNAs). It works in nucleolus and synthesis rrna 5,8S, 18S, 28S. - RNA-polymerase II works in nucleoplasme, synthesis mrnas and snrnas (10-40%). - RNA-polymerase III works in nucleoplasme, synthesis trnas and rrna 5S (10%). Transcription factors There are molecules (usually proteins) that mediate transcription by interaction with DNA or other proteins implicated in transcription. There are two types of transcription factors: General transcription factors are the same for all cell. Their functions: - Facilitate the interaction between promoter and RNA-polymerase - Participate in choosing of template strand and indicate direction of transcription - Unwind and rewind double helix of DNA - Prevent premature removing of RNA-polymerase from template - Assure termination of transcription. 2
Leading sequence Transcription and processing Specific transcription factors are specific for each kind of cells. They participate in decondensation of chromatin and bind specific to promoter, indicating Transcribed region Promoter Coding region Terminator Point of initiation of transcription Site of initiation of translation Site of termination of translation Site of polyadenilation the active one. Fig. 2. Structure of gene coding mrna in eukaryotes Particularities of transcription of mrna in eukaryotic cell Initiation Fig. 3. Initiation of transcription Fig. 2 represents the structure of gene coding for mrna. For initiation of transcription there are some events, which consist in activation of gene and beginning of transcription: - Decondesation of chromatin. Demethylation of DNA. - Interaction of specific factor of transcription with promoter. - Binding of TFIID (TBP) to TATA-box (TF Transcription Factor, II polymerase II; TBP TATA Binding Protein); - TFIIA binds upstream from the TBP. It stabilizes the complex TBP-TATA-box.; - In front of TBP binds TFIIB, which unwind DNA using ATP; - RNA-polymerase II, activated by TFIIF binds to promoter. TFIIF is a helicase, which unwind locally DNA; - After binding of factors TFIIE and TFIIH RNA-polymerase can move along the template strand of DNA; - Reading of first nucleotide from 3
Transcription and processing template (+1) end incorporation of first ribonucleotide, usually ATP; - Initiation is finished by formation of first phosphodiester bond in newly synthesized RNA (Fig. 3). Some distant sequences can also participate in the process of transcription. They may facilitate the recognition of promoter by RNA-polymerase (enhancer) or interfere in this process (silencer) (Fig. 4) Regulatory protein attached to enhancer Regulatory protein attached to promoter Enhancer Promoter Enhancer Promoter RNA polymeraze II Fig. 4. Interaction of enhancer (via regulatory proteins) with promoter and RNA-polymerase Elongation - From RNA-polymerase are released TFIIB and TFIIE. TFIIF and FTIIH remain attached to enzyme. - RNA polymerase II reads DNA in direction 3' 5' and polymerizes RNA in direction 5' 3' (30 bases/sec) - TFIIS prevents premature removing of RNA-polymerase - At the promoter remain attached: FTIID, FTIIA that can interact with other RNApolymerase. Fig. 5. Structure of terminator Termination In Fig. 5 is shown the structure of terminator. It contains a palindromic sequence a region in which the sequence on both strands is identical when read in an antiparallel direction. After RNA-polymerase transcripts the sequence corresponding to the terminator RNA forms a hairpin loop. RNApolymerase stops and a factor of termination rho (ρ) interact with enzyme and dissociate the complex DNA-enzyme-RNA (Fig. 6). 4
Transcription and processing RNA-polymerase transcribes DNA rho attaches to recognition site on RNA rho moves along RNA, following RNApolymerase RNA-polymerase pauses at terminator and rho catches up; rho unwinds DNA-RNA hybrid Termination: RNA-polymerase, rho and RNA are released Fig. 6. Termination of transcription Processing of RNA The primary transcript represents an immature RNA. Processing of RNA represents the events when the ends of RNA are modified and the non-coding sequences are removed from the pre RNA (Fig. 7). Fig. 7. The steps of processing of RNA 5
CAPing Transcription and processing The 5'-end of primary transcript is modified during transcription. After 30 bases were synthesized, guanilat-trasferase adds a methylated GTP by unusual bond 5'-5' to the first nucleotide of RNA (usually an Adenine). This structure ( 7 MeG 5 ppp 5 N) is named CAP. Also can be methylated the next riboses in position 2' (Fig. 8). CAP has the next functions: Stabilizes RNA due to unusual bond 5' - 5'; Represents a site of recognition for ribosome during initiation of translation. Polyadenylation After transcription the 3'-end of RNA contain a palindromic loop, which is removed by excision in the site AAUAAA. The enzyme poly(a)- Fig. 8. The structure of CAP polymerase adds 100-200 residues of adenilic acid. mrna which cods for histones are not polyadenylated. Poly(A)-tail has some functions: Assures the stability of 3'-end of RNA. Molecules that contain more long tails are more stabile. Participates in passing of mrna thru nuclear envelope. Splicing Splicing represents the process of removing of introns from pre mrna and sealing of exons. This process takes place with participation of an enzymatic complex splicesome. Enzymes (U 1 -U 6 ) represent ribonucleoproteins and contain snrna. Introns are recognized by sequences GU at 5'-end and AG at 3'-end. There are some steps in the process of splicing (Fig. 9): Site GU is recognized by U 1 ; U 2 binds to an Adenine from the interior of intron (branch site); U 4,U 5,U 6 associate to U 1 and U 2 forming a loop by binding of 5'-end of intron to Adenine via an unusual bond 5' 2'; After removing of U 4 3'-end of intron is cleaved. The intron forms a lasso and is removed together with proteins U 2, U 5, U 6 ; The 3' and 5'-end of exons are sealed. Fig. 9. Splicing pre-mrnas and assembly of spliceosomes Resulting mrna is transported to the cytoplasm, where will be used as template for protein synthesis. There are some types of splicing: Constitutive splicing all introns are removed from pre mrna and exons are sealed in the same consecution as in gene. Alternative splicing in mrna remain only some of exons, and only some of introns are removed. From one gene can be synthesized more types of proteins. 6
Transcription and processing Exon shuffling change of consecution of exons in mrna from the position in gene. Trans-splicing exons from different pre mrna participate to make one molecule of mrna. RNA editing RNA editing is defined as a process responsible for any differences between the final sequence of a messenger RNA (mrna) and its genetically determined template. This process takes place in cytoplasm and involves adding, removing or conversion of some nucleotides. Particularities of I -st and II -nd class genes transcription Eukaryotic ribosomes contain four RNA molecules: 5S, 5,8S, 18S and 28S. In the nucleolus there are several hundred copies of transcription units which encod for 5,8S, 18S and 28S. The mammalian primary transcript of I-st class genes is a 45S RNA containing the sequences of 5,8S, 18S and 28S rrnas. During maturation, the primary transcript is cleft in 5 places, spacers are Fig 10. Transcription and processing of rrna removed and 3 types of rrna are made (Fig 10). The genes for the 5S rrna is contained in a separate transcription unit. These genes are also arranged in tandem: there are several repeating units separated by untranscribed segments (Fig. 11). This type of rrna is transcribed by RNA-polymerase III. Note that in higher eukaryotes the rrna sequences do not contain introns. Fig. 11. Organization of genes for the 5S rrna Eukaryotic trna molecules are also excised from large transcripts (called pre-trna), which may contain one or more trna sequences. During processing the introns and spacer sequences are removed, at the 3 end a specific CCA sequence is added. Transcription in mitochondria In mammalian mitochondrial genomes are very compact; they are no introns. The 13 mrna, 2 rrna and 22 trna genes are under control of two promoters: HSP and LSP. Most genes are expressed in the same direction and trna genes lie between the genes coding for rrna or protein. 12 mrna-coding, 2 rrna-coding and 14 trna-coding regions are transcribed in clockwise direction; 1 mrna-coding and 8 trna-coding regions are read counter clockwise. Beginning with 7
the promoter DNA is transcribed into a single transcript, from which RNAs are cleaved to release the mrnas, rrnas and trnas. Transcription and processing Transcription in prokaryotes In bacteria and other prokaryotes, several genes may be grouped together to form a single transcription unit under the control of a promoter an operon. In an operon, genes encoding, for example, the different enzymes of a metabolic pathway or the subunits of an enzyme complex, are clustered and are transcribed together into a polycistronic transcript, under the control of a single promoter. This transcript is then translated to give the individual proteins. Operons enable the rapid and efficient coordinate expression of a set of genes required to respond to a change in the external or internal environment (See Fig. 6 THE GENE). 8