SIBC504: TRANSCRIPTION & RNA PROCESSING Assistant Professor Dr. Chatchawan Srisawat

TRANSCRIPTION: AN OVERVIEW Transcription: the synthesis of a single-stranded RNA from a doublestranded DNA template. The first stage in the overall process of gene expression. The most commonly-used step in regulating gene expression in organisms. Transcriptional control Switch gene on or off Phenotype

TRANSCRIPTION: AN OVERVIEW Important features of transcription RNA polymerase is the key enzyme: it requires DNA template, ribonucleotide precursors (ATP, GTP, CTP, and UTP) It catalyzes the synthesis of RNA from ribonucleotide precursors using one of the two DNA strands as a template. Coding or sense strand Template or antisense strand The sequence of the RNA transcript is the same as that of the coding strand (with U in place of T).

TRANSCRIPTION: AN OVERVIEW Important features of transcription Coding or sense strand Template or antisense strand Direction of synthesis occurs from the 5 - to the 3 -end of the RNA.

TRANSCRIPTION: AN OVERVIEW Important features of transcription Transcription process can be divided into three steps: 1. Initiation upstream -1 +1 downstream RNA polymerase transcription start site promoter A region of DNA involved in binding of RNA polymerase to initiate transcription; usually located upstream to the start site. Binding of RNA polymease to specific DNA sequences called promoters. Local unwinding of DNA and synthesizing the first few nucleotides of RNA (no primers required).

TRANSCRIPTION: AN OVERVIEW Important features of transcription Transcription process can be divided into three steps: 2. Elongation Successive addition of ribonucleotides into the RNA transcript from 5 to 3 direction using DNA as a template. DNA unwinding ahead of RNA polymerase and reannealing behind the enzyme.

TRANSCRIPTION: AN OVERVIEW Important features of transcription Transcription process can be divided into three steps: 3. Termination Synthesis of the RNA transcript is stopped at the terminator sequence. RNA polymerase dissociates from the DNA and releases the RNA transcript.

TRANSCRIPTION INITIATION Transcription initiation upstream -1 +1 downstream RNA polymerase promoter transcription start site The first step of transcription process Regulation of gene expression at transcriptional level are the most commonly used mode to determine which genes to be active or inactive. Most of regulatory mechanisms of gene expression occur at the transcriptional initiation step.

TRANSCRIPTION INITIATION IN PROKARYOTE In prokaryotes, RNA polymerase is a multisubunit enzyme. RNA polymerase holoenzyme Core enzyme σ (sigma) factor alpha (α) -2subunits/enzyme - required for core protein assembly; may be involved in promoter binding beta (β) - involved in catalysis; chain initiation and elongation. beta (β ) - involved in template DNA binding. omega (ω) - promotes assembly. Core enzyme can catalyze RNA synthesis and bind to DNA, but has no specificity; unable to start transcription at initiation sites alone.

TRANSCRIPTION INITIATION IN PROKARYOTE In prokaryotes, RNA polymerase is a multisubunit enzyme. RNA polymerase holoenzyme Core enzyme σ (sigma) factor Responsible for binding of DNA at promoters. Bring the core RNA polymerase enzyme to the initiation sites (promoters) to initiate transcription. Required for initiation and released from the core enzyme at the start of transcription elongation.

TRANSCRIPTION INITIATION IN PROKARYOTE σ factors and promoters Many bacteria, including E. coli, produce a set of different σ factors. σ 70, σ 32, σ 28, σ 38, σ 54 Number denotes the molecular weight of σ factor. σ 70 is the most common σ factor found in E. coli.

TRANSCRIPTION INITIATION IN PROKARYOTE σ factors and promoters upstream downstream promoter Promoter A region of DNA involved in binding of RNA polymerase to initiate transcription; usually located upstream to the start site. Different promoters contain conserved sequences, which can be specifically recognized and bound by different σ factors of RNA polymerase, thus facilitating assembly of the enzyme at the transcription initiation site.

TRANSCRIPTION INITIATION IN PROKARYOTE σ 70 factor and its promoter sequence The σ 70 promoter consists of a sequence of between 40-60 bp (region from around -55 to +20), which is bound by RNA polymerase. consensus sequence of σ 70 promoter -35 sequence TTGACA Recognition region interacting with σ factor -10 sequence or Pribnow box TATAAT 16-18 bp 5-8 bp CGT Important for DNA unwinding G A +1 Note - Different σ factors recognize specific promoters with different conserved sequences.

TRANSCRIPTION INITIATION IN PROKARYOTE σ 70 factor and its promoter sequence

TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation Promoter binding RNA polymerse core enzyme The RNA polymerase core enzyme has a nonspecific affinity for DNA, making it difficult and very slow to search and bind to the promoter by itself. With σ factor, the affinity for nonspecific sites on DNA is reduced by 20000-fold, while the affinity for promoters is enhanced by 100-fold. σ factor increases the specificity of the enzyme for promoter-binding sites.

TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation Promoter binding At the promoter, the RNA polymerase recognizes the promoter sequences at -35 and -10 regions (via the interaction with σ factor) Formation of the initial complex of the RNA polymerase and the basepaired DNA at the promoter: a closed complex.

TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation DNA unwinding Around 12 bp of the DNA (from -9 to +3) is unwound by the polymerase, forming an open comlex. -10 sequence region (AT-rich) is important for DNA unwinding

TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation RNA chain initiation -35 sequence TTGACA -10 sequence or Pribnow box TATAAT 16-18 bp 5-8 bp CGT G A +1 RNA polymerase starts synthesizing a short RNA chain without the movement of the enzyme from the promoter. The synthesis does not require a primer. A short RNA chain of 9 nt in length is generated; the first base is usually started with G or A. The process is ineffective. It may abort and then restart until initiation succeeds.

TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation RNA chain initiation When initiation succeeds, the enzyme releases the σ factor (which is not required for elongation) and forms a ternary complex of polymerase-dna-rna transcript. Transcription elongation begins as the enzyme moves along the DNA to synthesize RNA transcript.

TRANSCRIPTION INITIATION IN PROKARYOTE Alternative sigma factors Sigma factors confer promoter specificity to the core RNA polymerase. The presence of alternate sigma factors provides the cell with a mechanism for turning on and off entire families of genes under different circumstances. Note- Deviation of a promoter sequence from the consensus may affect the promoter strength and efficiency of transcription

TRANSCRIPTION INITIATION IN EUKARYOTE In eukaryotes, there are many kinds of RNAs with different functions. There are also three different kinds of RNA polymerases (Pol I, II, and III) to transcribe these RNAs in eukaryotic cells.

TRANSCRIPTION INITIATION IN EUKARYOTE Eukaryotes have 3 types of RNA polymerases responsible for transcription of nuclear genes RNA polymerase I transcribes rrna RNA polymerase II - transcribes mrna (all protein-coding RNA) RNA polymerase III - transcribes trna, 5S RNA, snrna, 7SL RNA Distinguished by sensitivity to α-amanitin In addition, there are organelle-specific RNA polymerases (i.e., mitochondria and chloroplast) to transcribe genes in the organelles.

TRANSCRIPTION INITIATION IN EUKARYOTE Comparison of structures of eukaryotic and prokaryotic RNA polymerases Eukaryotic RNA polymerase (Pol I, II, and III) are multimeric enzymes and some of the subunits are conserved, showing similarity to the prokaryotic counterparts. Note- the carboxy terminus (CTD) of β like subunit of RNA pol II contains repeats of seven amino acids YSPTSPS - (up to 50 repeats), which can be phosphorylated during transcription.

TRANSCRIPTION INITIATION IN EUKARYOTE Typical RNA polymerase II core promoter Core promoter: the minimal DNA region at which RNA polymerase II can bind and initiate basal transcription of the gene. 1. TATA box - located approximately 25-30 bp upstream of the start site - the conserved sequence: 5 TATA A A T A T 3 2. Initiator element (Inr) - usually located at start site - the conserved sequence: 5 Py 2 CAPy 5 3

TRANSCRIPTION INITIATION IN EUKARYOTE Transcription initiation of RNA polymerase II Like the core enzyme of RNA polymerase in prokaryotes, the RNA polymerases in eukaryotes also cannot bind to the promoter by itself. General or basal transcription factors are required for RNA pol II to initiate transcription at the promoter.

TRANSCRIPTION INITIATION IN EUKARYOTE Transcription initiation of RNA polymerase II General or basal transcription factors Not subunits of the RNA polymerase II. Required for RNA polymerase to bind avidly and specifically to promoters. General transcription factors for RNA polymerase II are called TFIIx, where x = A, B, D,... Can have multiple subunits.

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 1. TFIID assembles on the TATA box - possibly stimulated by TFIIA TFIID - a multisubunit complex One subunit called TBP (TATAbinding protein) and other components called TAF ll s (TBP-associated factors) Only TBP binds to DNA (at TATA box) in sequence specific manner

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 1. TFIID assembles on the TATA box - possibly stimulated by TFIIA TBP binds to TATA box and bends the DNA ~80 o, forcing open the minor groove. Helps unwind the DNA and may allow interaction with distant elements. rasmol

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 1. TFIID assembles on the TATA box - possibly stimulated by TFIIA TFIIA enhances and stabilizes the binding of TFIID to the TATA box. Probably stops inhibitory factors binding to TFIID, which could otherwise block further assembly of the transcription complex.

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 2. Binding of TFIIB to the preinitiation complex. TFIIB binds to TFIID and acts as a bridge for RNA polymerase binding.

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 3. RNA pol II is escorted to the preinitiation complex along with TFIIF. RNA polymerase II is in a complex with TFIIF. TFIIF delivers the RNA polymerase to the TFIID- TFIIA-TFIIB complex (via interactions with TFIIB). Could be involved in melting DNA at initiation.

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 4. TFIIE, TFIIJ, and TFIIH are then recruited to the preinitiation complex. TFIIE acts as docking site for TFIIH and can stimulate transcription. TFIIJ functions are stil unknown.

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 4. TFIIE, TFIIJ, and TFIIH are then recruited to the preinitiation complex. TFIIH a complex protein with 9 subunits Has DNA helicase/atpase activity for melting DNA during transcription initiation. Also has kinase activity: phosphorylates the carboxyl terminal domain (CTD) of the large β like subunit of RNA polymerase II

TRANSCRIPTION INITIATION IN EUKARYOTE Sequential model of initiation 5. Phosphorylation of C-terminal domain of RNA pol II by TFIIH. TFIIH phosphorylates the CTD of of the large β like subunit of RNA polymerase II. formation of a processive RNA polymerase complex. allows the RNA polymerase to leave the promoter region to begin transcription elongation. also serves as Landing pad for macromolecular assemblies that regulate mrna synthesis and processing, e.g., transcriptional elongation factors, RNA modifications enzymes.

TRANSCRIPTION INITIATION - SUMMARY Promoter binding of RNA polymerases prokaryote eukaryote Pol III RNA polymerase I promoters RNA polymerase II promoters RNA polymerase III promoters

TRANSCRIPTION ELONGATION RNA polymerase moves along DNA, unwinding it, exposing 10-20 DNA bases to be used as a template -> transcription bubble Adds to the 3 end (grows from 5 to 3 direction of RNA transcript) As it passes, DNA helix rewinds and new RNA peels away. Add 60 nucleotides/sec in eukaryotes, 40 nucleotides/sec in prokaryotes.

TRANSCRIPTION ELONGATION In eukaryotes, TFIIF remains associated with RNA polymerase II through out transcription elongation. During elongation, the activity of RNA polymerase II are greatly enhanced by proteins called elongation factors: eg., ELL, p- TEFb, SII(TFIIS), Elongin (SIII). The elongation factors suppress pausing or arrest of transcription and also coordinate interactions between protein complexes involved in the posttranscriptional processing of mrnas. Once the RNA transcript is completed, transcription is terminated. Pol II is dephosphorylated and recycled, ready to initiate another transcription.

Termination in prokaryotes TRANSCRIPTION TERMINATION There are two ways to terminate transcription in E.coli ρ (rho)-dependent termination ρ (rho)-independent termination

Termination in prokaryotes TRANSCRIPTION TERMINATION ρ (rho)-dependent termination Some genes contain terminator sequences which require an additional protein factor, ρ, for efficient termination. ρ binds to specific sites in singlestranded RNA. Then it hydrolyzes ATP and moves along the RNA towards the transcription complex, enabling the polymerase to terminate transcription.

Termination in prokaryotes TRANSCRIPTION TERMINATION ρ (rho)-independent termination Elongation of RNA transcript continues until the RNA polymerase reaches the terminator sequence. The most common terminator is an RNA hairpin in which the RNA transcript is self-complementary.

Termination in prokaryotes TRANSCRIPTION TERMINATION ρ (rho)-independent termination RNA polymerase pauses immediately after it has synthesized the hairpin RNA. The subsequent stretch of U s basepairs only weakly with da s in the DNA template strand. Release of the RNA transcript. The two DNA strands reanneal. Dissociation of the core enzyme from the DNA.

Termination in eukaryotes TRANSCRIPTION TERMINATION Termination of RNA polymerase I and III transcription Both RNA polymerases, I and III, have discrete termination sites (like prokaryotic RNA polymerase). RNA polymerase I terminates at a discrete site, involving a recognition region of an 18-base terminator sequence by an ancillary factor. RNA polymerase III terminates at the sequence with a run of 4 U bases without the need of any accessory factors.

Termination in eukaryotes TRANSCRIPTION TERMINATION Termination of RNA polymerase II RNA polymerase II elongates past the cleavage site at AAUAAA (polyadenylation or cleavage signal) and ceases RNA synthesis within multiple termination sites located in a rather long terminator region. Sometimes >1000 bp downstream of the 3 end The nature of the individual termination sites and sequences is not known because the mature 3 end of the mrna is generated by cleavage at a specific sequence, not by the termination event.

RNA PROCESSING Messenger RNA processing in prokaryotes no significant modification Messenger RNA processing in eukaryotes 5 -capping Splicing 3 -cleavage and polyadenylation

RNA PROCESSING 5 -CAPPING Functions: Marks the 5 -end of the first exon and aids in the splicing process Essential for nucleo-cytoplasmic transport of mrnas through interaction with nuclear cap-binding proteins Increases the efficiency of translation by targeting formation of the preinitiation complex (cytoplasmic cap-binding proteins) protects the transcript from 5 3 exoribonucleolytic activities

5 -CAPPING RNA PROCESSING

RNA PROCESSING 3 -CLEAVAGE AND POLYADENYLATION An enzyme complex recognizes the polyadenylation or cleavage signal (AAUAAA) and a less well conserved G-U rich sequence located 20-40 nucleotides downstream. An endonuclease cleaves the primary transcript 10-30 nucleotides downstream of the AAUAAA signal. A series of 80-250 A residues are added to the 3 -end of the cleaved transcript by polyadenylate polymerase.

RNA PROCESSING 3 -CLEAVAGE AND POLYADENYLATION Functions: Increase translational efficiency Protection of mrna from degradation

RNA PROCESSING SPLICING Two steps of transesterifications The 2 -OH from an adenine in intron attack 5 splicing site. 3 -OH at the 5 exon attach the 3 splicing site, joining the two exons together. Intron is excised as a lariat.

RNA PROCESSING SPLICING The chemistry is simple, but the in vivo process in not. Need to accurately define where exons end and introns begin. Catalysis of splicing reactions. SPLICEOSOME A large RNA/protein complex consists of specialized RNA-protein complexes, small nuclear ribonucleoproteins (snrnps). U1, U2, U4, U5, and U6 snrnp Each snrnp contains one of a class of eukaryotic RNAs, 100 to 200 nucleotides long, known as small nuclear RNAs (snrnas).

RNA PROCESSING SPLICING Recognition of exon/intron boundaries Exon - Ex pressed Sequences Intron - In tervening Sequences Polypyrimidine tract (U or C) consensus sequence for 5 splice site (donor site) consensus sequence for 3 splice site (acceptor site) Most introns start from the sequence GU and end with the sequence AG (in the 5' to 3' direction); the splice donor (5 splice site) and splice acceptor (3 splice site), respectively. In over 60% of cases, the exon sequence is (A/C)AG at the donor site, and G at the acceptor site. Another important sequence is called the branch site located 20-50 bases upstream of the acceptor site; "CU(A/G)A(C/U)", where A is conserved in all genes.

RNA PROCESSING SPLICING Splicing mechanism in mrna primary transcripts

RNA PROCESSING

Cotranscriptional pre -mrna processing RNA PROCESSING

RNA PROCESSING