BCM II LC 6 Transcription PDF

BIOCHEMISTRY LC 6: TRANSCRIPTION DR. BRENDO V. JANDOC | 01/28/2025 - COURSE OUTLINE...

BIOCHEMISTRY LC 6: TRANSCRIPTION DR. BRENDO V. JANDOC | 01/28/2025 - COURSE OUTLINE A. RNA POLYMERASE I.​ TRANSCRIPTION ​ RNA polymerase incorporates incoming A.​ RNA Polymerases ribonucleotides into a growing RNA strand by 1.​ Reaction Catalyzed forming phosphodiester bonds with the pre-existing 2.​ Prokaryotes RNA. B.​ Messenger RNAs (mRNAs) C.​ Other RNAs REACTION CATALYZED D.​ Important Features of Transcription ​ (RNA)n residues + NTP ←→ (RNA)n+1 residues + PPi 1.​ Highly selective ​ (RNA)n: Existing RNA strand w/ n nucleotides 2.​ Posttranscriptional modification ​ NTP (Nucleoside Triphosphate): A building block of E.​ Untranslated Regions RNA (ATP, UTP, CTP, or GTP). F.​ AUG (Methionine) Codon ​ (RNA) ₊₁: The elongated RNA strand, now with one II.​ STEPS IN RNA SYNTEHSIS additional nucleotide. A.​ Template Recognition ​ PPᵢ (Inorganic Pyrophosphate) → A byproduct of 1.​ Polymerase-binding site the reaction, which is later hydrolyzed to drive the 2.​ Promoter sequences reaction forward. a)​ Nomenclature and numbering ​ Mechanism: conventions ​ RNA polymerase binds to DNA and facilitates the b)​ Consensus sequences in the incorporation of NTPs complementary to the DNA promoter region template strand. c)​ Prokaryotic promoters ​ A phosphodiester bond is formed between the d)​ Eukaryotic Promoters 3’-OH of the growing RNA chain and the B.​ Initiation α-phosphate of the incoming NTP. 1.​ Initiation Factors ​ This results in the elongation of the RNA strand by 2.​ Prokaryotic σ Factor one nucleotide. 3.​ Assembly of Eukaryotic General ​ Pyrophosphate (PPᵢ) is released, and its hydrolysis Transcription Factors by pyrophosphatase drives the reaction forward. 4.​ Role of Enhancers in Eukaryotic Gene Regulation PROKARYOTES 5.​ Inhibitors of RNA Polymerase II ​ In prokaryotes, only one species of RNA 6.​ RNA Polymerase I and III polymerase synthesizes all 3 types of RNA except C.​ Elongation for the short RNA. 1.​ Overview of RNA polymerase ​ It doesn't synthesize RNA primers as it is 2.​ Prokaryotic RNA Polymerase synthesized by the ribosomes containing the 3.​ Eukaryotic RNA Polymerase primase.​ ​ ​ D.​ Termination 1.​ Prokaryotes B. MESSENGER RNA (mRNAs) 2.​ Eukaryotes ​ Transcripts of certain DNA regions, translated into E.​ Transcription Units sequences of amino acids (polypeptide chains) F.​ Determining Transcription Start Sites ​ Initial or Primary mRNA G.​ Footprinting ○​ A complete copy of one strand of DNA H.​ Action of Antibiotics ○​ Contains both introns and exons III.​ POST-TRANSCRIPTIONAL RNA ○​ Complementary to the DNA template (antisense) MODIFICATION strand and identical to the coding A.​ Prokaryotes (sense/antitemplate) strand (with U replacing T) 1.​ Messenger RNA ​ When mRNA is transcribed, the initial primary 2.​ Ribosomal RNA transcript is a complete copy of the DNA. It contains 3.​ Transfer RNA both coding regions and non-coding sequences, B.​ Eukaryotes including introns (also known as spacers) and 1.​ Ribosomal RNA inter-cistronic regions. 2.​ Messenger RNA ​ The initial or primary transcript in eukaryotes is a)​ Heterogenous Nuclear RNA called heterogenous nuclear RNA. b)​ Poly-A Tail Addition c)​ Splicing 3.​ Transfer RNA a)​ Large Precursor RNAs b)​ Introns IV.​ SUMMARY I. TRANSCRIPTION Figure 1. Relationship between the coding strand of DNA (sense ​ The conversion of genetic information of strand or the non-template strand), the DNA template strand (antisense strand), the mRNA transcript, and the protein produced polypeptides and proteins relies on the transcription from the gene, the bases in mRNA are used in sets of three (called of sequences of bases in DNA to messenger RNA codons) to specify the order of the amino acids inserted into the molecules. growing polypeptide chain during the process of translation. BATCH TANNAWAG 1E​ 1 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 C. OTHER RNAs ​ Heterogeneous nuclear RNA (hnRNA) undergoes ​ Includes: post-transcriptional modifications, such as capping, ○​ Ribosomal RNAs (rRNAs) splicing, and polyadenylation, to become the final ○​ Transfer RNAs (tRNAs) functional copy of mRNA ready for translation. ○​ Small RNA molecules ​ Processes include: ​ Perform specialized structural and regulatory ○​ Terminal additions functions without translation, so they are ​ Capping: at the 5’ end non-coding RNAs ​ Poly-adenylation: at the 3’ end ○​ Base modifications D. IMPORTANT FEATURES OF ○​ Trimming TRANSCRIPTION ○​ Internal segment removal ○​ Splicing HIGH SELECTIVITY ​ Introns are removed ​ Achieved by a small nuclear RNA in ​ Many transcripts are made of some regions of the association with specific proteins DNA; few or no transcripts are made from other ​ Alternative splicing is possible whereby an regions entire exon can be omitted → more than one ​ Due to: protein can be coded from the same gene ○​ Signals: embedded in the nucleotide sequences of the DNA which instructs the RNA polymerase Table 1. Types and function of post-transcriptional modification. where, how, and when to start transcription ○​ Regulatory proteins: like transcription factors ​ The high selectivity of transcription serves as the basis for the biochemical differentiation of an organism’s tissues. While all cells in the body contain the same DNA, different genes are selectively expressed in specific tissues. For example, the gene encoding thyroid hormone is expressed only in the thyroid gland, while the gene for erythropoietin is expressed in the kidneys. The E. UNTRANSLATED REGIONS thyroid gland does not express the erythropoietin ​ First 20 or more nucleotides gene because it is not the site of its production. ​ Contains recognition and regulatory sequences This selective gene expression ensures that each necessary for translation and possibly earlier tissue produces only the proteins necessary for its transcription function. ​ Poly A tail ○​ Not translated (3’ untranslated region) ○​ Preceded by stop codon (UAA, UAG, or UGA) ○​ Note that this is not a product of transcription but added after transcription Figure 3. Structure of eukaryotic mRNA. Untranslated regions both from the 5’ and 3’ end contain sequences that are recognition or regulatory sequences necessary for more efficient initiation of transcription. F. AUG (METHIONINE) CODON ​ Codon that will signal the start of translation II. STEPS IN RNA SYNTHESIS A. TEMPLATE RECOGNITION Figure 2. Expression of genetic information by transcription. High ​ Involves binding of RNA polymerase II to a specific selectivity of transcription implies that not all genes (boxes) are sequence of DNA molecules (DNA promoter transcribed (either only one, or two, or all, or even none at all). region) ​ The first step in RNA synthesis is template POST-TRANSCRIPTIONAL MODIFICATION recognition. The enzyme must gain access to the ​ While still in the nucleus, mRNA undergoes DNA and bind to the appropriate template modifications to convert the inactive primary sequence. transcript into a functional, mature mRNA. BATCH TANNAWAG 1E ​ 2 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 resulting mRNA is complementary to the template strand but is an exact copy of the coding (non-template) strand, except U replaces T. In gene mapping, anything downstream (to the right) is labeled as positive (+), while anything upstream (to the left) is labeled as negative (-), indicating relative positions from the transcription start site. Figure 4. Template Recognition in Transcription. RNA polymerase II binds to the DNA double helix at the promoter region, initiating transcription by unwinding the DNA for template recognition. B. Consensus Sequences in the Promoter Region ​ do not tolerate mutational changes, remain POLYMERASE-BINDING SITE constant even in evolutionarily remote organisms ​ defines the starting point of transcription (the sequences are conserved in evolution) ​ bacterial RNA polymerase binds to a specific region ​ mutations at different sites upstream of a gene have of about 60 base pairs of the DNA different effects depending on where they are ​ Start of the transcription located ​ Promoter sequences are critical regions in DNA that serve as binding sites for enzymes and transcription factors to initiate transcription. Because these sequences play an essential role in gene expression, they do not tolerate mutations well. Any significant change could disrupt transcription, leading to faulty or no protein production. Therefore, these consensus sequences are highly conserved through evolution, meaning Figure 5. Polymerase binding site. they remain almost identical across different species to ensure proper gene regulation PROMOTER SEQUENCES ​ transcription does not require a primer ​ responsible for directing RNA polymerase to initiate transcription at a particular point ​ differ between prokaryotes and eukaryotes ​ DNA sequence that specifies the site of RNA polymerase binding from which transcription is initiated Figure 7. Effect of promoter region on the rate of transcription. ​ organized into several regions with sequence homology PROKARYOTIC PROMOTERS ​ This will dictate or tell the enzyme where to bind ​ contain “consensus” nucleotide sequence of six base pairs TATAAT ​ highly conserved; many different promoters contain some very similar or identical sequences Figure 6. Promoter of transcription. A. Nomenclature and Numbering Conventions ​ used to avoid confusion in the description of sequences Figure 8. Structure of the prokaryotic promoter region. ​ sequence of only 1 DNA strand is presented ​ RNA synthesis occurs in a 5’ to 3’ direction 3 Sequence Elements sequences are written in a 5’ to 3’ direction ​ Initiation Site (Start point / Transcription Start ​ sequence of the DNA strand that is identical to the Site) RNA transcript is presented ○​ most gene transcriptions always start at the same ​ position 1 of a gene is the base that is equivalent to base (position 1) the 1st base of the 5’-end of the RNA transcript of ○​ usually a purine that gene ​ TATAAT or Pribnow or -10 Box ​ sequences preceding the 1st base ○​ stretch of 6 nucleotides (TATAAT) ○​ numbered negatively ○​ exists about 9-18 base pairs upstream of the start ○​ said to be upstream of the initiation point point (10 base pairs above the starting point) ​ sequences following the 1st base ○​ has been called the -10 sequence (minus 10 ○​ numbered positively sequence) because it is usually found 10 base ○​ said to be downstream of the initiation point pairs upstream of the start point ​ 1st base at the transcription start site is assigned a ○​ mutation at this region can affect gene position of +1 transcription controlled by the promoter ​ DNA and RNA are always synthesized and read in ​ TTGACA or -35 Box the 5' to 3' direction, meaning new nucleotides are ○​ another region of conserved sequences, typically added at the 3' end. DNA consists of two found 35 base pairs upstream of the start point antiparallel strands, where the lower strand is the ○​ also recognized by RNA polymerase template (antisense) strand used for mRNA ○​ mutation at this region can affect gene synthesis. During transcription, T pairs with A, A transcription controlled by the promoter pairs with U (in RNA), and C pairs with G. The BATCH TANNAWAG 1E ​ 3 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Two Important Regulatory Sequences ​ usually followed by three or more adenine bases ​ -35 Box ​ sequence of DNA nucleotides that is almost ○​ 5’-TATTGACA-3’ identical to the Pribnow box ​ 10 Box (TATA Box) ​ binding site of RNA polymerase II and GTF ○​ 5’-TATAAT-3’ Location EUKARYOTIC PROMOTERS ​ -20-30 (25-35) bases upstream of the ​ each type of eukaryotic RNA polymerase uses a transcriptional start site different promoter ​ promoters used by RNA polymerase I and II, 5’-TATAAA-3’ upstream of the start point ​ core DNA sequence ​ promoters used by RNA polymerase III, usually downstream of the start point TATA Binding Protein ​ normally bind TATA box in the process of Initiation Site / Start Point transcription ​ promoters of all eukaryotic RNA polymerases direct ​ unwinds the DNA and bends it through 80o the initiation of transcription to a particular starting point AT-Rich Sequence ​ usually a purine (pyrimidine is not uncommon) ​ facilitates easy unwinding (due to 2 hydrogen bonds between bases as opposed to 3 between RNA POLYMERASE I GC pairs) - site of bonding ​ has a bipartite promoter - which synthesizes the Different Core Promoter Elements small RNAs ​ Majority of genes which have no TATA box. The ○​ one part 170 to 180 bp upstream (5’ direction) TATA box is absent in many eukaryotic genes, ○​ other from about 45 bp upstream to 20 bp which rely on alternative promoter elements for downstream (3’ direction) (core promoter) transcription initiation. ​ requires two ancillary factors ​ Inr (initiator) sequence: located at the transcription ○​ i. UPE1 (upstream promoter element 1) start site (+1) ○​ ii. SL1 ​ DPE (downstream promoter element): located ​ located in the nucleolus approximately +28 to +32 nucleotides downstream, ​ synthesizes ribosomal RNA (accounts for about serve as crucial recognition sites for the 50-70% of the relative activity) transcription machinery. ​ Synthesize 45s to give rise to 28, 18, and 5.8 ​ The Inr and DPE facilitate the binding of RNAs. transcription factor TFIID, which ensures the recruitment of RNA polymerase II for transcription. ​ GC-rich regions in promoters are more thermodynamically stable due to three hydrogen bonds per base pair (versus AT pairs’ two), making them more difficult to unwind. ​ This increased stability in GC-rich regions can reduce transcription efficiency as the transcription machinery needs more energy to separate the DNA Figure 9. RNA Polymerase I promoter. strands. RNA POLYMERASE II ​ requires a transcription factor complex (TFIID) that binds to a single upstream promoter ​ located in the nucleoplasm ​ represents 20-40 % of cellular activity ​ responsible for the synthesis of heterogeneous Figure 10. Eukaryotic gene promoter consensus sequences. nuclear RNA (hnRNA) which is the precursor of mRNA ​ Also synthesize, noncoding RNAs. 2. CAAT Box and CG Box ​ 2nd consensus sequence; distinct pattern of nucleotides with a GGNCAATCT sequence ​ occurs 50-130 (40-200) bases upstream of the initial transcription site ​ signals the binding site for the RNA transcription factor Figure 10. RNA Polymerase II promoter. ​ typically accompanied by a conserved sequence ​ genes that have this element seem to require it for RNA POLYMERASE II PROMOTERS transcription in sufficient quantities 3. Cis-Acting Genetic Elements 1. TATA or Hogness Box (Goldberg Hogness Box) ​ term for the promoter DNA sequences that are on ​ a consensus sequence (nearly the same in all the same DNA molecule or chromosome as the organisms); almost identical to the pribnow box genes being transcribed ​ highly conserved in evolution ​ binding site for general transcription factors ​ about 4-8 base pairs BATCH TANNAWAG 1E ​ 4 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 4. Trans-Acting Genetic Elements INITIATION FACTORS ​ transcription factors encoded by a gene on ​ Needed to initiate transcription different chromosome ​ Prokaryotes ​ ​ synthesized in the cytosol ○​ Only a single factor is needed which is the sigma ​ can diffuse through the cell to points of action factor including to different chromosomes ​ Eukaryotes ​ stimulate or inhibit transcription of particular genes ○​ Multiple factors are required ​ Factors produced by the other chromosomes PROKARYOTIC σ FACTORS RNA POLYMERASE III ​ required for accurate initiation of transcription ​ uses either upstream promoters or two internal ​ Function promoters downstream of the transcription start site ○​ enables the RNA polymerase holoenzyme to ​ located in the nucleoplasm recognize and bind tightly to the promoter ​ responsible for the synthesis of sequences ​ tRNAs ​ other small RNAs ​ contributes only minor activity of about 10% Transcription Factors Required with Internal Promoters ​ TFIIIA - a zinc finger protein ○​ TFIIIB - a TBP and two other proteins ○​ TFIIIC - a large protein; > 500 kD Figure 13. Local unwinding of DNA caused by RNA polymerase. RNA POLYMERASE III PROMOTERS ​ downstream of the start point ​ Process ○​ Upon binding → opening or melting of DNA double helix phosphodiester bond formation between the 1st 2 bases (1st base is usually a purine nucleoside triphosphate: pppA or pppG) → 10 nucleotides added → factor σ dissociates → core enzyme continues to elongate the transcript ○​ Released factor → combine with free core Figure 11. RNA polymerase III promoter. 5S rRNA Promoter enzyme → holoenzyme formation → able to ​ made of 2 sequence elements in the transcribed initiate transcription portion of the gene ​ Upon binding it will cause separation of the 2 ○​ sequence 50-70 base pairs downstream of the strands of the DNA and then duplicate the template start point synthesizing mRNA from the 5’ to 3’ until you have ○​ sequence 80-90 base pairs downstream of the 10 nucleotides added, and the sigma factor will start point dissociated and then the beta sub-unit of the holoenzyme will continue elongating the mRNA. tRNA Promoters ​ In the figure the mRNA is complementary base pair ​ made of 2 sequence elements to the template, so you have a DNA RNA binding. ○​ sequence between +8 and +30 ○​ sequence between +50 and +70 ASSEMBLY OF EUKARYOTIC GENERAL ​ downstream of the start point within the transcribed TRANSCRIPTION FACTORS portion of the gene ​ The general transcription factors (interact with each other and with RNA polymerase II) and B. INITIATION RNA Polymerase II are needed to initiate ​ Opening of the DNA double helix → initiation transcription from TATA box promoters. complex makes the template strand available for base pairing. ​ Begins with the synthesis of the first RNA molecules at the initiation complex. ​ The RNA polymerase remains at the promoter while it synthesizes the first nine nucleotide bonds. ​ Requires several other proteins (activators and Figure 14. Transcription apparatus. The TATA-binding Protein (TBP), transcription factors). component of TFIID bonds to the TATA box. Transcription Factor IID (TFIID) ​ Transcription factor D for polymerase II ​ Recognizes and binds to the TATA box sequences independently of RNA polymerase II Figure 12. Initiation. ​ TATA-Binding Protein (TBP) ○​ Small; 30-kD; recognizes TATA Box ○​ Part of one of the many subunits of TFIID BATCH TANNAWAG 1E ​ 5 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Transcription Factor IIA (TFIIA) ROLE OF ENHANCERS IN EUKARYOTIC GENE ​ Associated with TFIID REGULATION ​ Binds to TATA box upstream of TFIID​ Enhancers ​ special cis-acting DNA sequences Transcription Factor IIB (TFIIB) ​ Function: ​ Binds to RNA polymerase II and TATA box ○​ increase the rate of initiation of transcription by downstream of TFIID ​ ​ RNA polymerase II when bound by transcription ​ Facilitates binding of RNA polymerase II to TFIID factors and TFIIA complex to the TATA box ​ Location ​ Is an ATPase, which cleaves ATP upon formation of ○​ On the same chromosome as the gene whose the preinitiation complex of RNA polymerase II, transcription they stimulate TFIID, TFIIA, and TFIIB ○​ “Upstream” (to the 5′-side) or “downstream” (to the 3′-side) of the transcription start site Transcription Factor IIE (TFIIE) ○​ Close to or thousands of base pairs away from ​ Binds to the preinitiation complex the promoter ​ Transcription begins at the start point in the ○​ On either strand of the DNA presence of ribonucleoside triphosphates ​ Response Elements ○​ DNA sequences contained by enhancers TFIIH ○​ Bind specific transcription factors (activators) ​ Escorted by TFIIF ○​ Increase the rate of transcription. ​ Ensure that Pol II is attached to the promoter ​ Other activities: ​ Helicase ​ ATPase Polymerase II ​ Activated by phosphorylation à transcription begins va. Polypeptide Tail ​ Site of phosphorylation ​ Composed in mammals of 52 repeats of the amino acid sequence YSPTSPS ​ Serine (S) and threonine (T) side chains are phosphorylated ​ Transcription factors IIA, IIB, IIE, IIH, IIF, and TFIIB recognize and binds to the TATA sequence accompanied by TFIID, TFIIH, TFIIE, and THIIF. ​ TFIIF upon binding will guide the RNA Pol II to position itself along the DNA. ​ In summary, TFIIB binds to TATA supported by the different transcription factors and the enzyme will Figure 16. Enhancer stimulation of RNA polymerase II. be guided by TFIIF to position itself in a proper position in the DNA at the promoter region. ​ The same transcription factors will phosphorylate the enzyme to activate the enzyme. (Enzymes activated by phosphorylation and deactivated by dephosphorylation). Figure 17. Some possible locations of enhancer sequences. Figure 15. Assembly of General Transcription Factors. BATCH TANNAWAG 1E ​ 6 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 ​ Silencers ​ Negative supercoils behind it - will be relaxed ○​ act over long distances by topoisomerases I and Ii ○​ reduce the level of gene expression when bound ​ Requirements ○​ Opposite the function of enhancers ○​ Same in eukaryotes and prokaryotes ​ Template INHIBITORS OF RNA POLYMERASE II ○​ Single strains of DNA ​ Substrate α-Amanitin ○​ 4 ribonucleoside triphosphate: ATP, GTP, CTP, ​ Enzyme; potent toxin UTP ​ Produced by the poisonous mushroom Amanita ○​ Cleavage of the high energy phosphate bond phalloides (“death cap” or “destroying angel”) between alpha and beta phosphates provides the ​ Forms a tight complex with the polymerase → energy for the addition of nucleotides to the inhibition of mRNA synthesis and protein synthesis growing RNA chain → energy (no active polymerase, no active transcription, no ​ Direction of Synthesis protein synthesis) ○​ Except for the 1st nucleoside triphosphate ○​ Subsequent nucleotides are added to the 3’hydroxyl of the preceding nucleotide (5’ → 3’ direction) - the same principle in the DNA replication PROKARYOTIC RNA POLYMERASE ​ Properties of Prokaryotic RNA Polymerase ○​ 1 species of RNA polymerase for all cellular RNA synthesis except for the short RNA primers needed for DNA replication Figure 18. α-Amanitin ○​ Multisubunit enzyme ○​ Recognize and bind to a nucleotide sequence (promoter region) at the beginning of the length of DNA to be transcribed ○​ Makes complementary RNA copy of the DNA Figure 19. Summary of the types of RNA Polymerases. template ○​ Recognize the termination region RNA POLYMERASE I AND III ○​ RNA is synthesized from its 5’ → 3’ end (antiparallel to its DNA template) ​ Also need specific transcription factors to initiate ○​ Transcription Unit transcription from their respective promoters ​ Extends from the promoter to the termination ​ Utilize different transcription factors but they follow region the same principle. ○​ Primary transcript ​ Product of the process of transcription by C. ELONGATION RNA polymerase ​ Promoter region recognition by holoenzymes → synthesis of DNA sequence transcript by RNA 1. RNA Polymerase Holoenzyme in E. Coli polymerase with release of σ subunit ​ Required for proper initiation of transcription ​ Begins when the enzymes move along the DNA → ​ Binds DNA relatively weakly except at specific extend the RNA chain promoter sequences recognized by the σ subunit ​ DNA helicases aid in the process. The helicase will continue to unwind to further free the portion of the Components of Holoenzyme: template synthesizing the mRNA from the 5’ to 3’ i.​ Core Enzymes leading the DNA from the 3’ to the 5’ direction ○​ Required for the elongation steps of RNA synthesis OVERVIEW OF RNA POLYMERASE ○​ Peptide subunits ​ RNA Polymerase ​ 2 α: 40k MW ○​ Does not require a primer because they are ​ 1 β: 155k MW already RNA ​ 1 β’: 160k MW ○​ No known Endo- or exonuclease activities → no ○​ 5’ - > 3’ RNA polymerase activity ability to repair mistakes, which means it is error ○​ Lacks specificity (cannot recognize the promoter prone but if there is misincorporation, the RNA region of the DNA template) polymerase can pause and cleave the ○​ Binds DNA tightly misincorporated segment and then restart again ○​ Half-life of approx. 60 min ○​ Utilizes ribonucleoside triphosphates, which ii.​ σ Subunit (Sigma Factor) release pyrophosphate each thime a nucleotide ○​ 85k MW is added to the growing chain ○​ Enables the polymerase to recognize the ○​ Binding with the DNA template → DNA helix promoter region of the DNA template unwinding ○​ Different σ factors recognize different groups of ○​ As it pushes its way between the strands of genes; different species have different sigma double helix → creates: factor ​ Positive supercoils ahead of the transcription site - will be relaxed by gyrase BATCH TANNAWAG 1E ​ 7 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 ○​ Dissociates from the complex on the initiation of Table 2. Eukaryotic RNA polymerase and their Functions. transcription, leaving the core enzyme to continue transcription 2. Termination Factor ​ Some DNA regions that signal the termination of transcription are recognized by the RNA polymerase itself ​ Others are recognized by specific termination factors -​ Ex: Rho (p) Factor of E. Coli EUKARYOTIC RNA POLYMERASE D. TERMINATION ​ RNA polymerase is removed from the DNA ​ Mitochondrial RNA Polymerase ​ The formation of the unstable primary transcript is ○​ Single species only complete ○​ Resembles bacterial polymerase more closely ​ Immediately translated into prokaryotes than it does the eukaryotic enzyme ​ Modified (processed) in eukaryotes ​ Nuclear RNA Polymerase ​ Transcription ends as RNA polymerase detaches; ○​ Synthesize different RNAs the primary transcript is immediately translated in ○​ Structure: Large enzymes > 500,000 MW with > prokaryotes but undergoes modification in 10 subunit polypeptides eukaryotes. ○​ Location: Subnuclear localization Classes of Eukaryotic Nuclear RNA Polymerase ​ Large enzymes with multiple subunits ​ Each unit recognizes particular types of genes 1. RNA Polymerase I Figure 20. Termination. ○​ Located in the nucleolus, which synthesizes the precursor of large ribosomal RNAs in the PROKARYOTES nucleolus 1.​ρ-Independent Termination -​ 28S ​ Particular sequences can cause the core enzyme to -​ 18S terminate transcription -​ 5.8S ​ Required structural features of the newly ○​ mRNA and tRNA synthesized RNA molecule -​ synthesized in the nucleoplasm ​ Requires that a sequence in the DNA template generate a sequence in the nascent RNA that is 2. RNA Polymerase II self-complementary ○​ Located in the nucleoplasm, which synthesis ​ RNA polymerase halts when it encounters a precursors of mRNAs and small nuclear RNA hairpin-forming GC-rich region that is followed by a (snRNA) series of U residues on the DNA sense strand ○​ Used by some viruses to produce viral RNA ○​ General transcription factors are required for simple basal transcription of most class II genes ○​ Promoter for Class II Genes: may serve as recognition sites for eukaryotic promoters a.​TATA or Goldberg - Hogness Box b.​CAAT Box: ○​ RNA polymerase inhibitors: ​ α-Aminitin: bicyclic octapeptide; potent toxin by mushroom Amanita phalloides (called death cap or destroying angel), forms a tight complex with the polymerase → mRNA synthesis inhibition Figure 21. Rho(ρ)-independent termination of prokaryotic transcription. DNA template sequence generates a → protein synthesis inhibition self-complementary sequence in the nascent RNA. 3. RNA Polymerase III Stable Hairpin Turn -​ Located in the nucleoplasm, which produces ​ Slows down the progress of RNA polymerase and the small RNAs (i.e., tRNAs. 5S rRNA, and pauses temporarily some snRNAs) ​ Complementary to a region of the DNA template near the termination region that exhibits twofold symmetry (due to presence of a palindrome) ​ A stable hairpin turn forms in the RNA transcript, causing RNA polymerase to slow down and pause temporarily. This structure arises from a palindromic sequence in the DNA template, where both strands have identical nucleotide sequences when read in the same direction. BATCH TANNAWAG 1E ​ 8 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 ​ Palindrome: 2. ρ-Dependent Termination ○​ Region of double-stranded DNA. Each of the 2 ​ ρ - Factor strands contain stretches that have the same ○​ Hexameric ATPase nucleotide sequence when read in the same ○​ Binds to a C-rich “rho recognition site” near the direction 3’-end of the nascent RNA -> migrates until it ​ Guanosine-Cytosine (G-C)-Rich Stem-Loop reaches the RNA polymerase paused at the Base termination site in the 5’->3’ direction -> ○​ Important in the termination of transcription ATP-dependent RNA-DNA helicase activity of rho ○​ Very stable G-C base pairs separates the RNA-DNA hybrid helix -> RNA ○​ Makes the formation of a stem-loop structure release ○​ Favorable or more stable ○​ The rho (ρ) protein binds to a C-rich recognition site near the 3′ end of the RNA and moves along it in the 5′→3′ direction until it reaches RNA polymerase at the termination site. Using ATP-dependent helicase activity, rho unwinds the RNA-DNA hybrid, leading to the release of the RNA transcript. EUKARYOTES ​ Few identified termination sequences ​ Transcription continues for up to several thousand base pairs beyond the point of polyadenylation Figure 22. The recognition sequence of restriction endonuclease E. TRANSCRIPTION UNITS EcoRI shows twofold rotational symmetry. dsDNA = double-stranded ​ Segment of DNA between the sites of initiation and DNA; A = adenine; C = cytosine; G = guanine; T = thymine. termination of transcription String of Us ​ Following the hairpin turn ​ Bonding of U to the corresponding DNA template A is weak -> facilitates separation of the newly synthesized RNA from the DNA template ​ Termination becomes favorable when the RNA polymerase pauses ​ No specific base where transcription stops Figure 25. Transcription unit. Figure 23. Rho(ρ)-independent termination of prokaryotic transcription. The RNA forms the hairpin structure. “N” represents a noncomplementary base. [NOTE: termination of eukaryotic transcription is not well understood] Figure 24. Typical prokaryotic termination sequence. Figure 26. Transcription by RNA polymerase II. BATCH TANNAWAG 1E ​ 9 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 F. DETERMINING TRANSCRIPTION START RIFAMPIN SITES ​ Rifampicin derivative ​ Bind to the β-subunit of the prokaryotic RNA 1. Nuclease S1 Protection Assay polymerase (when the polymerase is in the ​ Used to analyze a segment of DNA suspected to holoenzyme form) -> interference with the formation contain a transcription start site of the 1st phosphodiester bond -> inhibition of the initiation of transcription 2. Endonuclease S1 ​ These molecules bind to the β-subunit of ​ Enzyme present in the mold Aspergillus oryzae prokaryotic RNA polymerase in its holoenzyme ​ Cleaves single-stranded DNA and RNA but not form, preventing the formation of the first double-stranded phosphodiester bond. This inhibition blocks the ​ All single-stranded DNA will be removed allow initiation of transcription, halting RNA synthesis. relevant DNA to be identified ​ No effect on eukaryotic nuclear RNA polymerases ​ Treatment of tuberculosis Figure 27. Determining transcription start sites. G. FOOTPRINTING ​ A technique used to identify the promoter region ​ RNA polymerase and DNA (containing the putative promoter) -> incubated with alkylating reagent at DNA bases that are not protected by contact with the RNA polymerase -> cleavage of the DNA backbone at the alkylated residues -> products Figure 29. Inhibition of prokaryotic RNA polymerase by rifampin. analyzed by electrophoresis to determine the region of DNA that binds to RNA polymerase DACTINOMYCIN (ACTINOMYCIN D) ​ To identify the promoter region, RNA polymerase is ​ Binds to DNA template → interferes with RNA incubated with DNA and an alkylating reagent, polymerase movement along the DNA which modifies unprotected DNA bases. Cleavage ​ Tumor chemotherapy at these modified sites followed by electrophoresis reveals the DNA region that binds RNA polymerase. III. POST-TRANSCRIPTIONAL RNA MODIFICATION 1. Primary Transcript or Heterogeneous Nuclear RNA (hnRNA) ​ Linear copy of a transcriptional unit ​ RNA is immediately produced by RNA polymerase (before it undergoes modification). 2. Transcriptional Unit ​ DNA segment between specific initiation and termination sequences 3. Primary Transcripts of tRNAs and rRNAs Figure 28. DNA footprinting (via electrophoresis) ​ Post-transcriptionally modified by cleavage of the original transcripts by ribonucleases H. ACTION OF ANTIBIOTICS ​ Some antibiotics prevent cell growth through RNA 4. Prokaryotic mRNA synthesis inhibition ​ Generally identical to its primary transcript (no modification) BATCH TANNAWAG 1E ​ 10 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 5. Eukaryotic mRNA TRANSFER RNAs (tRNAs) ​ Extensively modified post-transcriptionally ​ Large Precursor Transcripts ○​ Give rise to tRNAs not formed from processing ​ tRNA Genes ○​ Clustered, containing sequences for 2-7 tRNAs ​ Cleavage by: ○​ ribonuclease P ○​ ribonuclease D ○​ removal of the portions of the transcript that form functional tRNAs ​ Addition of Sequence -CCA to the 3’-End ○​ by tRNA nucleotidyl transferase ○​ CCA sequence is common to all tRNAs ​ Base Modification Figure 30. Transcription unit. ○​ Either by methylation or more extensive Table 3. Factors that affect modification of the primary transcript. modifications ○​ production of unusual bases necessary for the tRNAs to adopt their unique, functional conformations B. EUKARYOTES ​ RNAs are processed during their transport from the nucleus to cytosol needed to be functional in the cytoplasm allowing for another level of gene regulation A. PROKARYOTES RIBOSOMAL RNAs (rRNAs) MESSENGER RNAs (mRNAs) ​ not post-transcriptionally processed ​ functional immediately after synthesis ​ translation often begins before transcription is complete RIBOSOMAL RNAs (rRNAs) ​ Preribosomal RNAs ​ long precursor molecules of ribosomal RNAs ​ 7 Genes Produce rRNA ​ each gene produces 30S precursor rRNA which is processed into functional rRNA ​ containing sequences that become: Figure 30. the eukaryotic ribosomal RNA (rRNA) processing ○​ 23S rRNA pathway, showing transcription, modification, and assembly of ribosomal subunits in the nucleolus before their export to the ○​ 16S rRNA cytoplasm for functional ribosome formation. ○​ 5S rRNA ​ some of the tRNA genes are within the transcribed 1. Preribosomal (45S) RNAs portion. ​ precursor molecule with no tRNA sequences ​ different rRNA genes contain different tRNA genes. ​ made by the transcription of hundreds of separate ​ Cleavage by: rRNA genes in the nucleolus by RNA polymerase I ○​ ribonuclease P ​ Highly methylated before it is cleaved to the ○​ ribonuclease III functional rRNAs ​ non-functional spacer sequences are removed ​ spacer sequences are removed by endonucleolytic ​ Base Modification cleavage by specific endonucleases ○​ by methylation → functional rRNAs ​ 5.8S rRNA base pairs with the 28S rRNA during the formation of the ribosomal subunits ​ Yields ○​ 28S rRNA ○​ 18S ○​ 5.8S 2. 5S rRNA ​ Synthesized by the transcription of 5S gene by RNA polymerase III Figure 29. Processing of 30S Precursor rRNA by RNase P and RNase III - the maturation of prokaryotic rRNA. The 30S precursor ​ modified separately rRNA contains 16S, 23S, and 5S rRNA along with tRNA and spacer sequences. Enzymes RNase P and RNase III cleave the precursor, releasing the functional rRNA subunits (16S, 23S, and 5S) and tRNA, which are essential for ribosome assembly and protein synthesis. BATCH TANNAWAG 1E ​ 11 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Possible Cap Structures ​ Depending on the presence or absence of additional methyl groups on the 2 nucleosides adjacent to the 7-methyl guanylate I.​ Cap 0: When there is no additional methylation beyond the methylated guanosine. II.​ Cap 1: When the 1st ribose sugar adjacent to the 7-methylguanylate is methylated. Figure 31. The processing of eukaryotic 45S precursor rRNA, III.​ Cap 2: When the 1st and 2nd ribose sugars highlighting the cleavage of spacer sequences by specific adjacent to the 7-methyl guanylate are endonucleases to produce mature 18S, 5.8S, and 28S rRNA methylated components for ribosome assembly. Figure 32. The transcription of the ribosomal RNA gene by RNA polymerase I, producing pre-rRNA, which is then processed by RNases to generate the mature 18S, 5.8S, and 28S ribosomal RNAs. MESSENGER RNAs (mRNAs) ​ Formed from extensive processing of hnRNA A. Heterogeneous Nuclear RNA (hnRNA) ​ Primary RNA transcript molecule synthesized by Figure 33. 5' and 3' modifications of eukaryotic mRNA, including the RNA polymerase II 7-methylguanosine cap structure (Cap0, Cap1, and Cap2) at the 5' end and the poly-A tail at the 3' end, which are essential for mRNA ​ Contains the sequences that are found in cytosolic stability and translation. mRNA ​ Extensively modified after transcription which may B. Poly-A Tail Addition (Polyadenylation) include:​ ​ ​ Necessary for all hnRNAs to be successfully ○​ C5’ “Capping”​ ​ converted to mRNA ​ 1st of the processing reactions for hnRNA ​ Chain of 200-300 adenine nucleotides attached to ​ begins during transcription or immediately the 3’-end thereafter ​ Not transcribed from the DNA (template ​ all mRNAs are capped independent) ​ added after transcription by nuclear polyadenylate Cap - 7-methylguanosine polymerase ​ attached backward through a triphosphate linkage ​ help stabilize mRNAs to the 5’-terminal of mRNA → unusual 5’→5’ ​ protects the mature mRNA from ribonuclease triphosphate linkage activity in cells ​ addition of guanosine triphosphate cap ​ facilitate their exit from the nucleus ○​ catalyzed by nuclear enzyme guanylyltransferase ​ gradually shortened after entering the cytosol ​ methylation of terminal guanine ​ endonuclease cuts the molecule on the 3′ side of ○​ occurs in the cytosol the sequence AAUAAA then poly-A polymerase ○​ catalyzed by guanine-7-methyl-transferase adds the poly-A tail ○​ S-adenosylmethionine as the methyl donor and ○​ additional methylation steps may occur Addition of 7-Methylguanosine Cap ​ facilitate more efficient initiation of translation and help to stabilize mRNA. ○​ Protection from digestion by ribonucleases that degrade RNAs from their 5’-end Figure 34. The structure of eukaryotic mRNA, highlighting the 5' (5’-exonucleases) 7-methylguanosine cap, the coding region for protein translation, the ​ lack of cap results in inefficient translation. polyadenylation signal sequence, and the 3' poly-A tail, all of which contribute to mRNA stability and translation efficiency. BATCH TANNAWAG 1E ​ 12 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Polyadenylation Signal Sequence (AAUAAA) Exons ​ consensus sequence ​ protein-coding sequence regions ​ near the 3’-end of the RNA molecule ​ expressed portions of the genes ​ signals that a poly-A tail is to be added to the ​ spliced together forming the mature mRNA mRNA ​ Occurs only after capping and before splicing Intron-Exon Junctions ​ all hnRNA introns have: a.) Guanine-Uracil (G-U) Signal That Identifies the Site of Polyadenylation Sequence - on the 5’ border, and b.) ​ lies within the RNA Adenine-Guanine (A-G) Sequence on the 3’ border Poly-A Polymerase ​ G-U and A-G sequences are flanked by ​ nuclear enzyme sequences that are identical to or similar in all ​ catalyzes the polymerization of adenylate residues introns onto the free 3’-end of the mRNA ​ Branch site within introns and conserved sequence Figure 35. Synthesis of poly (A) tail. As RNA polymerase continues to transcribe the DNA. enzymes cleave the transcript (hnRNA) at a point of 10 to 20 nucleotides beyond an AAUAAA sequence, just Figure 37. Eukaryotic gene expression process, showing before run if Us (or Gs). Approximately 250 adenine nucleotides are transcription, RNA splicing, and mRNA processing, where introns are then added to the 3’-end of the hnRNA, one at a time, by poly (A) removed via splice sites (GU-AG) and lariat formation, resulting in a polymerase. mature mRNA with a 5' cap and a poly-A tail for translation. Mechanism of Splicing ​ Catalyzed by spliceosome ​ Spliceosome ○​ Made up of large (50S to 60S) ribonucleoprotein complex ○​ Made of 5 snRNPs that contain 5 snRNAs ​ U1, U2, U4, U5, and U6 ○​ binding of snRNPs → bring together RNA sequences into perfect alignment for splicing ○​ 2’-hydroxyl group of an adenosine residue (branch site) in the intron. ​ attacks and forms a phosphodiester bond with Figure 36. Transcription and polyadenylation process in eukaryotic the phosphate at the 5’-end of the intron mRNA, where RNA polymerase initiates transcription, cleavage ​ newly-freed 3’-OH of the upstream exon occurs 11–30 nucleotides after the AAUAAA signal, and poly(A) polymerase adds a poly-A tail to the 3' end for mRNA stability and forms phosphodiester bond with the 5’-end of translation efficiency. the downstream exon C. Splicing ​ process of removing non-coding sequences or introns ​ all sequences necessary to form mRNA that codes for a protein product are contained in the hnRNA ​ coding sequences are often split or separated by non-coding sequences Introns ​ intervening RNA sequences between exons Figure 38. Spliceosome-mediated RNA splicing process, where small ​ do not code for proteins from the primary transcript nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6, along with proteins, ​ transcribed portions of the genes that are removed form small nuclear ribonucleoproteins (snRNPs) that recognize splice in the processing of hnRNA to mRNA sites, facilitate intron removal, and join exons to generate mature mRNA. ​ different genes have different numbers of introns of different sizes. ○​ β-Globin Genes: 2 introns ○​ LDL Receptor Gene: 17 introns BATCH TANNAWAG 1E ​ 13 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Figure 39. Spliceosome-mediated splicing process, where small nuclear ribonucleoproteins (snRNPs) recognize splice sites, facilitate the cleavage of introns at the GU and AG sequences, form a lariat structure, and ligate exons to generate mature mRNA. Figure 41. General and spliceosome-mediated splicing, showing the recognition of splice sites, the formation of a lariat intermediate at the branch site, and the sequential cleavage and ligation of exons to produce mature mRNA. Role of Small Nuclear RNAs (snRNAs) ​ in association with proteins forming small nuclear ribonucleoprotein particles (snRNPs) ​ for recognition of the conserved sequences in the introns ​ facilitate the splicing of some exon segments by forming base pairs with the consensus sequences at each end of the intron Systemic Lupus Erythematosus ​ often fatal inflammatory disease ​ results from autoimmune response in which the patient produces antibodies against snRNPs Effect of Splice Site Mutations ​ lead to improper splicing thus production of aberrant proteins ​ 15% of all genetic diseases are results of mutations that affect RNA splicing ​ some cases of β thalassemia (incorrect splicing of -globin mRNA) ​ If G is replaced by C in the splice site then it will not be anymore considered as a splice site. Regulation of Gene Expression at the Level of Transcription ​ Promoters are critical for the initiation of transcription and mutations may decrease the quantity of gene transcribed. ​ Enhancer ​ Silencer Transport ​ After intron removal → mature mRNA molecule → pores in the nuclear membrane → cystosol Figure 40. Removal of introns. Mechanism of spliceosome-mediated RNA splicing, where snRNPs facilitate the formation of a lariat structure through a 2’→5’ bond at the branch site, followed by exon ligation to produce mature mRNA. BATCH TANNAWAG 1E ​ 14 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Figure 44. Alternative splicing patterns in eukaryotic mRNA. Figure 42. Regulation of gene expression at multiple levels, including transcription, RNA processing (alternative splicing), mRNA translation (editing), and post-translational protein activity. Figure 45. Alternative RNA splicing, where different exon combinations in the primary RNA transcript result in distinct mRNA variants, leading to the production of either calcitonin in thyroid C cells or CGRP (calcitonin gene-related peptide) in the hypothalamus. Figure 43. Central dogma of molecular biology, depicting the processes of transcription, RNA splicing, RNA transport, translation by ribosomes, and polypeptide synthesis in eukaryotic cells. Alternative Splicing of mRNA Molecule ​ Important mechanism for generating multiple Figure 46. mRNA synthesis. Transcription produces hnRNA protein isoforms from a single gene (multiple (pre-mRNA) from the DNA template. hnRNA processing involves the variations of mRNA and its protein products in addition of 5’-cap and poly(A)tail and splicing to join exons and different tissues) remove introns. The product, mRNA, migrates to the cytoplasm, in ​ resulting proteins differ slightly in their amino acid which it will direct protein synthesis. sequence → small functional differences (often these differences are restricted to certain tissues) TRANSFER RNAs (tRNAs) ​ allows for a high degree of functional flexibility and evolutionary advantage. ​ ex: different types of muscle cells ○​ produce the same primary transcript from the tropomyosin gene ○​ different patterns of splicing in the different cell types → production of tissue-specific tropomyosin protein molecules Calcitonin Gene ​ the primary transcript for the calcitonin gene contains six exons > splicing > two types of mature mRNA ○​ Calcitonin: consisting of exons 1-4 (excluding exons 5 and 6) and produced in the thyroid Figure 47. Synthesis of transfer RNA (tRNA), including transcription, ○​ Calcitonin-Like Protein (Calcitonin intron removal, base modifications, and the addition of the CAA sequence at the 3' end, followed by export through the nuclear pore Gene-Related Product, CGRP): found in the into the cytoplasm for protein translation. hypothalamus and consisting of exons 1, 2, 3, 5, and 6 and excluding exon 4 BATCH TANNAWAG 1E ​ 15 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 ​ Large Precursor RNAs ​ Introns ○​ may contain 1 or more tRNA sequences​ ○​ may be contained by some tRNAs ○​ tRNA Sequences ○​ small (14-50 nucleotides) ​ excised from the precursor by specific ○​ no sequence homology in different tRNAs endonucleolytic cleavage ○​ Splicing is different from mRNA as enzymes ○​ CCA Sequence recognize characteristic features of the tRNA to ​ added to the 3’-end after the tRNA is cleaved identify the intron-exon junctions from the precursor ​ nucleotidyltransferase ○​ Extensive modification → adopt final functional structure Figure 48. Processing of prokaryotic 30S precursor rRNA, where RNase P and RNase III cleave spacer sequences to produce mature 16S, 23S, and 5S rRNAs, along with tRNA, for ribosome assembly. Figure 50. Cloverleaf structure of rRNA, highlighting key modified bases such as dihydrouridine (D), ribothymidine (T), and pseudouridine (Ψ), along with functional regions including the D loop, anticodon loop, variable loop, TΨC loop, and the 3' amino acid attachment site. Reference(s): ​ Jandoc, B. (January 2025). DNA Replication [PPT]. ​ Ferrier, D. R. (2014). Biochemistry. Lippincott Williams & Wilkins. Figure 49. Precursor transfer RNA transcript (pre-tRNA). Mature (functional) tRNA after posttranscriptional modification. Modified bases include D (dihydrouracil), pseudo uracil, and m, which means that the base has been methylated. IV. SUMMARY Figure 53. Eukaryotic transcription overview, BATCH TANNAWAG 1E ​ 16 BIOCHEM LC 6: TRANSCRIPTION​ Dr. JANDOC, B. 01/28/2025 Figure 52. Comparative summary of transcription and RNA processing in prokaryotic and eukaryotic cells, highlighting differences in gene structure, RNA polymerases, transcription initiation, mRNA synthesis, termination, post-transcriptional modifications, ribosomes, and tRNA structure. BATCH TANNAWAG 1E ​ 17