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  1. 1. ROLES OF RNA IN CELLS  RNA molecules can function as biological catalysts and may have been the first carriers of genetic information.  RNA is a polymer, consisting of nucleotides joined together by phosphodiester bonds.  Each RNA nucleotide consists of a ribose sugar, a phosphate, and a base. RNA contains the base uracil  It is usually single stranded, which allows it to form secondary structures.
  2. 2. All cellular RNA types are transcribed from DNARNA are synthesized that are complementary and antiparallel to the DNA template strand.
  3. 3. In most organisms, each gene is transcribed from a single DNA strand, but different genesmaybe transcribed from one or the other of the two DNA strands.
  4. 4. The ribonucleotide to be added at the 3’ end of a growing RNA strand is specifiedby base pairing between the next base in the template DNA strandand the complementary incoming ribonucleoside triphosphate(rNTP). A phosphodiester bond is formed when RNA polymerasecatalyzes a reaction between the 3’ O of the growing strand and theα phosphate of a correctly base-paired rNTP. RNA strands always are synthesized in the 5’- 3’ direction and are opposite in polarity to their template DNA strands.
  5. 5. The genetic code is a system of purines and pyrimidinesused to send messages from the genome to the ribosomes to direct protein synthesis. With a non-overlapping code, the reading frame advances three nucleotides at a time,and a mRNA segment is therefore read as three successive triplets, coding for amino acids.
  6. 6.  The genetic code are triplet , commaless, non- overlapping codons present in the nucleotide sequence of mRNA, as read in the 5’-3’ direction. Each codon specifies either an amino acid or a stop signal. There are 64 possible codons in mRNA, 61 code for amino acids, hence, degenerate and wobbling). TAA, TAG and TGA are the stop codons which do not have a corresponding tRNA. The genetic code is universal.
  7. 7. THE TRANSCRIPTION APPARATUS In bacterial RNA polymerase, the core enzyme consists of 4 catalytic subunits: 2 copies of alpha (α), a single copy of beta (β), and single copy of beta prime (β’). A 5th unit (ω) has been identified recently. (a) The regulatory subunit known as thesigma (σ) factor joins the core to form the holoenzyme, which is capable of binding to a promoter and initiating transcription. (b) The molecular model shows RNA polymerase (shown in yellow) binding DNA.
  8. 8. 1. The core enzyme catalyzes the elongation of the RNA molecule by the addition of RNA nucleotides. α2: The two α subunits assemble the enzyme and bind regulatory factors. ß: this has the polymerase activity which includes chain initiation and elongation. ß: binds to DNA (nonspecifically). ω: restores denatured RNA polymerase to its functional form in vitro. It has been observed to offer a protective/chaperone function to the β subunit.2. Once bound, the sigma factor increases RNA polymerase specificity for certain promoter regions, depending on the specific σ factor. That way, transcription is initiated at the right region. When not in use, RNA polymerase binds to low-affinity sites to allow rapid exchange for an active promoter site when one opens. RNA polymerase holoenzyme, therefore, does not freely float around in the cell when not in use.
  9. 9. Eukaryotes have several types of RNA polymerases,characterized by the type of RNA they synthesize: RNA polymerase I synthesizes a pre-rRNA 45S, which matures into 28S, 18S and 5.8S rRNAs which will form the major RNA sections of the ribosome. RNA polymerase II synthesizes precursors of mRNAs and most snRNA and microRNAs. This is the most studied type, and due to the high level of control required over transcription, a range of transcription factors are required for its binding to promoters. RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol. RNA polymerase IV synthesizes siRNA in plants. RNA polymerase V synthesizes RNAs involved in siRNA- directed heterochromatin formation in plants. There are RNA polymerase types in mitochondria and chloroplasts. There are RNA-dependent RNA polymerases involved in RNA interference.
  10. 10. Rifampicin inhibits prokaryotic RNApolymerases; α-Amanitin eukaryotic RNApolymerase II
  11. 11. A transcription unit is a piece of DNA thatencodes an RNA molecule and the sequences necessary for its proper transcription. Each transcription unit includes a promoter, an RNA-coding region, and a terminator.
  12. 12.  A promoter is a DNA sequence that is adjacent to a gene and required for transcription. Promoters contain short consensus sequences that are important in the initiation of transcription. Consensus sequence comprises the most commonly encountered nucleotides found at a specific location. In bacterial promoters, consensus sequences are found upstream of the start site, approximately at positions 10 & 35.
  13. 13.  In all species, transcription begins with the binding of the RNA polymerase complex (or holoenzyme) to a special DNA sequence at the beginning of the gene known as the promoter. Activation of the RNA polymerase complex enables transcription initiation, and this is followed by elongation of the transcript. In turn, transcript elongation leads to clearing of the promoter, and the transcription process can begin yet again. Transcription can thus be regulated at two levels: the promoter level (cis regulation) and the polymerase level (trans regulation). These elements differ among bacteria and eukaryotes.
  14. 14. TRANSCRIPTION IN PROKARYOTES In prokaryotic DNA, several protein-coding genes commonly are clustered into a functional region, an operon, which istranscribed from a singlepromoter into one mRNA encoding multiple proteins with related functions. Translation of a bacterial mRNA canbegin before synthesis of the mRNA is complete.
  15. 15. During initiation of transcription, RNA polymerase forms atranscription bubble and begins polymerization of rNTPs at thestart site, which is located within the promoter region. RNA polymerase moves along the template strand of the DNA in the 3’- 5’direction, and the RNA molecule grows in the 5’- 3’ direction. Once a DNA region has been transcribed, the separated strands reassociate into a double helix, displacingthe nascent RNA except at its 3’ end. The 5’ end of the RNAstrand exits the RNA polymerase through a channel in the enzyme. Termination occurs when the polymerase encounters a termination sequence (stop site).
  16. 16. INITIATION
  17. 17. Transcription is initiated at the start site, which, inbacterial cells, is set by the binding of RNA polymerase tothe consensus sequences of the promoter. Transcription takes place within the transcription bubble. DNA is unwound ahead of the bubble and rewound behind it.
  18. 18. ELONGATION During elongation, RNA polymerase binds to about 30 base pairs of DNA (each complete turn of the DNA double helix is about 10 base pairs). At any given time, about 18 base pairs of DNA are unwound, and the most recently synthesized RNA is still hydrogen- bonded to the DNA, forming a short RNA-DNA hybrid. This hybrid is probably about 12 base pairs long, even shorter. The total length of growing RNA bound to the enzyme and/or DNA is about 25 nucleotides.
  19. 19. TERMINATION Transcription ends after RNA polymerase transcribes 2 types ofterminator sequences: in rho-independent termination, a GC-rich sequence followed by several U residues forms a "brake" that will help release the RNA polymerase from the template. In rho-dependent termination, binding of rho to themRNA releases it from the template.
  20. 20. SUMMARY: PROKARYOTIC TRANSCRIPTION1.Transcription is a selective process; only certain parts of the DNA are transcribed.2.RNA is transcribed from single-stranded DNA. Normally, only one of the two DNA strands, the template strand, is copied into RNA.3.Ribonucleoside triphosphates (RNTPs), are used as the substrates in RNA synthesis. Two phosphates are cleaved from an RNTP, and the resulting nucleotide is joined to the 3’OH group of the growing RNA strand.
  21. 21. 5.RNA molecules are antiparallel and complementary to the DNA template strand.6.Transcription is always in the 5’-3’ direction, which means that the RNA molecule grows at the 3’ end.7.Transcription depends on RNA polymerase- a complex, multimeric enzyme which consists of a core enzyme capable of synthesizing RNA, and other subunits that may join transiently to perform additional functions.8.The core enzyme of RNA polymerase requires a sigma factor in order to bind to a promoter and initiate transcription.9.Promoters contain short sequences crucial in the binding of RNA polymerase to DNA; these consensus sequences are interspersed with nucleotides that play no known role in transcription.10.RNA polymerase binds to DNA at a promoter, begins transcribing at the start site of the gene, and ends transcription after a terminator has been transcribed.
  22. 22. The consensus sequences in promoters of three eukaryotic genes illustrate the principle that different sequences can be mixed and matched toTRANSCRIPTION yield aIN EUKARYOTES functional promoter.
  23. 23.  RNA polymerase I transcribes the rRNA precursor molecules. RNA polymerase I promoters have two key components: (1) the core element, which surrounds the start site and is sufficient to initiate transcription, and (2) the upstream control sequence, which increases the efficiency of the core promoter.
  24. 24. The basal transcription apparatus assembles at RNA polymerase I promoters.
  25. 25.  RNA polymerase II produces most mRNAs and snRNAs. The promoters of genes transcribed by RNA polymerase II consist of a core promoter and a regulatory promoter that contain consensus sequences. Not all the consensus sequences shown are found in all promoters.
  26. 26.  The typical promoter for RNA polymerase II has a short initiator sequence, consisting mostly of pyrimidines and usually a TATA box about 25 bases upstream from the start point. This type of promoter (with or without the TATA box) is often called a polymerase II core promoter, because for most genes a variety of upstream control elements also play important roles in the initiation of transcription.
  27. 27.  RNA polymerase III is responsible for the production of pre-tRNAs, 5SrRNA and other small RNAs. RNA polymerase III recognizes several different types of promoters. OCT and PSE are consensus sequences that may also be present in RNA polymerase II promoters.
  28. 28.  The promoters for RNA polymerase III vary in structure but the ones for tRNA genes and 5S rRNA genes are located entirely downstream of the startpoint, within the transcribed sequence. In tRNA genes, about 30-60 base-pairs of DNA separate promoter elements; in 5S rRNA genes, about 10-30 base-pairs promoter elements
  29. 29. General transcription factors and thepolymerase undergo a pattern of sequential binding to initiate transcription of nuclear genes. (1) TFIID binds to the TATA box followed by(2) the binding of TFIIA and TFIIB. (3) The resulting complex is now bound by the polymerase, to which TFIIF has already attached.
  30. 30. (4) The initiation complex is completed by the addition of TFIIE, and TFIIH. TFIIH helicase activity and its associated kinase complexreferred to as TFIIKphosphorylates the C-terminal domainof RNA polymerase largest subunit. (5) After its ATP- dependent phosphorylation, the polymerase can initiatetranscription at the startpoint.
  31. 31.  The TATA-binding protein (TBP) is a subunit of the TFIID and plays a role in the activity of both RNA polymerase I and III transcription. TBP is also essential for transcription of TATA-less genes. TBP differs from most DNA-binding proteins in that it interacts with the minor groove of DNA, rather than the major groove and imparts a sharp bend to the DNA. TBP has been highly conserved during evolution. When TBP is bound to DNA, other transcription- factor proteins can interact with the convex surface of the TBP saddle. TBP is required for transcription initiation on all types of eukaryotic promoters.
  32. 32. TERMINATIONIn many of the genes transcribed by RNA polymerase II, transcription can end at multiple sites located within a span of hundreds or thousands of base pairs.Termination is coupled to cleavage, which is carried out by a termination factor that associates with RNA polymerase I and III.This complex may suppress termination until the consensus sequence that marks the cleavage site is encountered.mRNA is cleaved by the complex 10 to 35 base- pairs downstream of a AAUAAA sequence (which acts as a poly-A tail addition signal).
  33. 33. Unlike rho, which binds to the newly transcribed RNA molecule, the termination factor for RNA polymerase I binds to a DNA sequence downstream of the termination site.RNA polymerase III transcribes a terminator sequence that produces a string of U’s in the RNA molecule, like that produced by the rho- independent terminators of bacteria.Unlike rho-independent terminators in bacterial cells, RNA polymerase III does not require that a hairpin structure precede the string of U’s.
  34. 34. SUMMARY: EUKARYOTIC TRANSCRIPTION Several types of DNA sequences take part in the initiation of transcription in eukaryotic cells. These promoter sequences generally serve as the binding sites for proteins that interact with RNA polymerase and influence the initiation of transcription. Promoters are adjacent to or within the RNA coding region and are relatively fixed with regard to the start site of transcription. Promoters consist of a core promoter located adjacent to the gene and a regulatory promoter located farther upstream. Other sequences, called enhancers, are distant from the gene and function independently of position and direction. Enhancers stimulate transcription.
  35. 35.  General transcription factors bind to the core promoter near the start site and, with RNA polymerase, assemble into a basal transcription apparatus. The TATA-binding protein (TBP) is a critical transcription factor that positions the active site of RNA polymerase over the start site. Transcriptional activator proteins bind to sequences in the regulatory promoter and enhancers and affect transcription by interacting with the basal transcription apparatus. Proteins binding to enhancers interact with the basal transcription apparatus by causing the DNA between the promoter and the enhancer to loop out, bringing the enhancer into close proximity to the promoter. The three RNA polymerases found in eukaryotic cells use different mechanisms of termination.
  36. 36. Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3’ end of the mature mRNA. An AAUAAA sequence located slightly upstreamfrom the proper 3’ end then signals that the RNA chain should be cleaved about 10-35 nucleotides downstream from the signal site, followed by addition of a poly-A tail catalyzed by poly(A) polymerase.
  37. 37.  A 5’ “cap” (a guanosine nucleotide methylated at the 7th position) is joined to the 1st nucleotide in an unusual 5’ -5’ linkage. The 5 cap has 4 main functions: o Regulation of nuclear export o Prevention of degradation by exonucleases o Promotion of translation o Promotion of 5 proximal intron excision During the capping process, the first two nucleotides of the message may also become methylated.
  38. 38.  The poly(A) tail is important for the nuclear export, translation and stability of mRNA. The tail is shortened over time and when it is short enough, the mRNA is enzymatically degraded. In addition to the 5’ cap and poly-A tail, mRNA in eukaryotes is first made as heterogeneous nuclear mRNA (or pre-mRNA), and then processed into mature mRNA through the splicing out of introns.
  39. 39. Restriction enzyme analysis has revealed the presenceof introns ineukaryotic DNA.
  40. 40. Hybridization of aeukaryotic mRNA molecule with a gene which has one intron will produce two single-strandedDNA loops where the mRNA hashybridized to the DNA template strand plus an obvious double- stranded DNAloop. The double- stranded DNA loop representsthe intron, which contains sequences that do not appear in the final mRNA.
  41. 41. Alternative splicing results in alternate forms of mRNA and proteins.
  42. 42. Distinct isoforms of individual domains of multidomain proteinsfound in higher eukaryotes often are expressed in specific cell types as the result of alternative splicing of exons The ≈75-kb fibronectin gene (top) contains multiple exons.The EIIIB and EIIIA exons (green) encode binding domains for specific proteins on the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts includes the EIIIA and EIIIB exons, whereas these exons are spliced out of fibronectin mRNA in hepatocytes. In this diagram, introns (black lines) are not drawn to scale; most of them are much longer than any of the exons.
  43. 43. Spliceosomes remove introns from pre-mRNA. The spliceosome is an RNA-protein complex that splicesintron-containing pre-mRNA in the eukaryotic nucleus.
  44. 44. In a stepwisefashion, the pre- mRNA assembles with the U1 snRNP, U2 snRNP, and U4/U6 and U5 snRNPs (along with some non- snRNP splicingfactors), forming a mature spliceosome.
  45. 45. The pre-mRNA is then cleaved at the5’ splice site and the newly released 5’ end is linked to an adenine (A)nucleotide located at the branch-pointsequence, creating a looped lariatstructure. Next the 3’splice site is cleaved and the two ends of the exon are joined together, releasing the intron for subsequent degradation.
  46. 46. Clinical Significance: Alternative and Aberrant Splicing Introns protect the genetic makeup of an organism from genetic damage by outside influences such as chemical or radiation, and increase the genetic diversity of the genome without increasing the overall number of genes. Abnormalities in the splicing process can lead to various disease states. Many defects in the β-globin genes are known to exist leading to β-thalassemias. Some of these defects are caused by mutations in the sequences of the gene required for intron recognition and, therefore, result in abnormal processing of the β-globin primary transcript. Patients suffering from a number of different connective tissue diseases exhibit humoral auto-antibodies that recognize cellular RNA-protein complexes. Patients suffering from systemic lupus erythematosis have auto- antibodies that recognize the U1 RNA of the spliceosome.
  47. 47. involves cleavage of multiple rRNAs from a common precursor. The eukaryotic transcription unit that includes the genes for the three largest rRNAs occurs in multiple copies and arranged in tandem arrays with non- transcribed spacers separate the units. Each transcription unit includes the genes for the three rRNAs and transcribed spacer regions. The transcription unit is transcribed by RNA polymerase I into a single long transcript (pre-rRNA) with a sedimentation coefficient of about 45S. RNA processing yields mature rRNA molecules. RNA cleavage actually occurs in a series of steps which varies in order with the species and cell type but the final products are always the same three types of rRNA molecules.
  48. 48. : every tRNA gene is transcribed as a precursor that must be processed into a mature tRNA molecule by the removal, addition and chemical modification of nucleotides. Processing for some tRNA involves: o removal of the leader sequence at the 5’ end o replacement of two nucleotides at the 3’ end by the sequence CCA (with which all mature tRNA molecules terminate) o chemical modification of certain bases o excision of an intron The mature tRNA is often diagrammed as a flattened cloverleaf which clearly shows the base pairing between self-complementary stretches in the molecule.
  49. 49. Long double-stranded RNAs  Upon introduction,(dsRNA) occur naturally in cells. the long dsRNAs with complementary sequence of a part of the target gene, enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway  The dsRNAs get processed into 20-25 nucleotide by an RNase III-like enzyme called Dicer.
  50. 50.  The siRNAs assemble into endoribonuclease containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process.  Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA.  The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene.......geneticsvideosRNAi.wmv silencing.
  51. 51. are single-stranded RNA molecules containing about 22 nucleotides and thus about the same size as siRNAs.  These are generated by the cleavage of larger precursors using Dicer.  They function as post- transcriptional regulators of gene expression.  They act by either destroying or inhibiting translation of several mRNAs, usually by binding to a region of complementary sequence in the 3-UTR region of the mRNA.