Transcription Md. Habibur Rahaman (HbR) Dept. of Biochemistry and Microbiology North South University

Central Dogma of Life

Transcription The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.

Similarity between Replication and Transcription Both processes use DNA as the template. Phosphodiester bonds are formed in both cases. Both synthesis directions are from 5´ to 3´.

Differences between Replication and Transcription template double strands single strand substrate dNTPs NTPs primer yes no Enzyme DNA polymerase RNA polymerase product dsDNA ssRNA base pair A-T, G-C A-U, T-A, G-C

Differences between Replication and Transcription During replication, all the nucleotides in the DNA template are copied, but, during transcription, only small parts of the DNA molecule—usually a single gene or, at most, a few genes—are transcribed into RNA. Because not all gene products are needed at the same time or in the same cell, it would be highly inefficient for a cell to constantly transcribe all of its genes. Furthermore, much of the DNA does not code for a functional product, and transcription of such sequences would be pointless. Transcription is, in fact, a highly selective process—individual genes are transcribed only as their products are needed (Spatiotemporal: proper time and place)

X Transcription: How is an RNA strand synthesized? Regulated by gene regulatory elements within each gene. DNA unwinds next to a gene. RNA is transcribed 5’ to 3‘ Similar to DNA synthesis, except: NTPs instead of dNTPs (no deoxy-) No primer No proofreading Adds Uracil (U) instead of thymine (T) RNA polymerase X

Types of RNAs

Types of RNAs and Function

Different types of RNA, each encoded by different genes: mRNA Messenger RNA, encodes the amino acid sequence of a polypeptide. 2. tRNA Transfer RNA, transports amino acids to ribosomes during translation. 3. rRNA Ribosomal RNA, forms complexes called ribosomes with protein, the structure on which mRNA is translated. 4. snRNA 5. miRNA/siRNA Small nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing (e.g., exon splicing and intron removal). Micro RNA/small interfering RNA, short ~22 nt RNA sequences that bind to 3’ UTR target mRNAs and result in gene silencing.

Template The whole genome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the development requirement, physiological need and environmental changes. DNA regions that can be transcribed into RNA are called structural genes.

Template The template strand is the strand from which the RNA is actually transcribed. The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein.

Asymmetric transcription Only the template fragment is used for the transcription, but not the coding fragment However, both strands can be used as the templates. The transcription direction on different strands is opposite. This feature is referred to as the asymmetric transcription.

Asymmetric transcription coding strand template strand template strand coding strand

Asymmetric transcription In most organisms, each gene is transcribed from a single strand, but different genes may be transcribed from different strands

Organization of coding information in the adenovirus genome

A Gene is a Transcription Unit A transcription unit includes a promoter, an RNA-coding region, and a terminator.

Transcription in Prokaryotes Only a single RNA polymerase In E.coli, RNA polymerase is 465 kD complex, with 2 , 1 , 1 ', 1  subunit function  Determine the DNA to be transcribed  Catalyze polymerization  Bind & open DNA template  Recognize the promoter for synthesis initiation

E. coli RNA polymerase Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the  subunit of RNA-pol, and inhibit the RNA synthesis.

Transcription of Prokaryotes Initiation phase: RNA-pol recognizes the promoter and starts the transcription. Elongation phase: the RNA strand is continuously growing. Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

Prokaryotic promoter Consensus sequence

Reaching A Consensus Consensus sequences: Strongest promoters match consensus Up mutation: mutation that makes promoter more like consensus Down Mutation: virtually any mutation that alters a match with the consensus

Reaching A Consensus

Reaching A Consensus These sequences (-10 and -35) are on the non-template strand (coding strand/sense strand) and read 5’3’, left to right.

Initiation RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex. The unwound region is about 171 bp.

RNA-pol (2) - DNA - pppGpN- OH 3 The first nucleotide on RNA transcript is always purine triphosphate. GTP is more often than ATP. The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed. The three molecules form a transcription initiation complex. RNA-pol (2) - DNA - pppGpN- OH 3

No primer is needed for RNA synthesis. The  subunit falls off from the RNA-pol once the first 3-5 phosphodiester bond is formed. The core enzyme moves along the DNA template to enter the elongation phase.

Prokaryotic Transcription Initiation

Sigma Recognizes the -10 and -35 Sequences The structure of yeast RNA polymerase reveals a groove that may be used by DNA as it moves relative to the polymerase and a potential groove for the newly formed RNA. Mol Bio_Clark_Chapter: 06

Promoter Strength The three important regions : (i) the 35 consensus region (ii) the 10 consensus Pribnow sequence (iii) the initiation site. Nucleotide composition (AT-rich regions separate more easily than GC-rich regions) Degree of supercoiling Interference of topoisomerases The conformation of the DNA

Facts to know The start site itself is not marked by a consensus sequence but often has the sequence CAT The position of the start site is determined not by the sequences located there but by the location of the consensus sequences If the consensus sequences are artificially moved upstream or downstream, the location of the starting point of transcription correspondingly changes.

Elongation The release of the  subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 3 end Free NTPs are added sequentially to the 3 -OH of the nascent RNA strand.

RNA-pol, DNA segment of ~18nt and the nascent RNA form a complex called the transcription bubble The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out the transcription bubble as the synthesis is processing This rate of RNA synthesis is much lower than that of DNA synthesis

Transcription bubble About 8 nucleotides of newly synthesized RNA are paired with the DNA-template nucleotides at any one time

RNA-pol of E. Coli

RNA-pol of E. Coli As the transcription apparatus moves down the DNA template, it generates positive supercoiling ahead of the transcription bubble and negative supercoiling behind it

The termination occurs in either  -dependent or  -independent manner RNA polymerase must stop synthesizing RNA The RNA molecule must be released from RNA polymerase The newly made RNA molecule must dissociate fully from the DNA RNA polymerase must detach from the DNA template Transcription ends after the terminator has been transcribed, like a car that stops only after running over a speed bump. The termination occurs in either  -dependent or  -independent manner

-independent termination The termination signal is a stretch of 30-40 nucleotides on the RNA transcript, consisting of many GC followed by a series of U. The sequence specificity of this nascent RNA transcript will form particular stem-loop structures to terminate the transcription. The adenine–uracil base pairings downstream of the hairpin are relatively unstable and the formation of the hairpin may itself destablize the DNA–RNA pairing

-dependent termination The  factor, a hexamer, is a ATPase and a helicase

Summary Transcription is a selective process RNA is transcribed from single-stranded DNA NTPS are used as the substrates in RNA synthesis RNA molecules are antiparallel and complementary to the DNA template strand Transcription depends on RNA polymerase—a multimeric enzyme The core enzyme requires a sigma factor in order to bind to a promoter and initiate transcription Promoters contain short sequences crucial in the binding of RNA polymerase Transcription ends after a terminator has been transcribed

Transcription of Eukaryotes Differ from prokaryotes Eukaryotic cells possess three different RNA polymerases, each of which transcribes a different class of RNA and recognizes a different type of promoter Another difference is in the nature of promoter recognition and initiation. Many proteins take part in the binding of eukaryotic RNA polymerases to DNA templates, and the different types of promoters require different proteins.

Transcription of Eukaryotes

Transcription of Eukaryotes Need to gain access to DNA-Histone complex Acetyltransferases add acetyl groups to amino acids at the ends of the histone proteins, which destabilizes the nucleosome structure and makes the DNA more accessible Proteins called chromatin-remodeling proteins may bind to the chromatin and displace nucleosomes from promoters and other regions important for transcription

Transcription of Eukaryotes Transcription initiation needs promoter and upstream regulatory regions. The cis-acting elements are the specific sequences on the DNA template that regulate the transcription of one or more genes.

Cis-acting element

Transcription factors RNA-pol does not bind the promoter directly. RNA-pol II associates with six transcription factors, TFII A - TFII H. The trans-acting factors are the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.

Transcription factors General transcription factors, which, along with RNA polymerase, form the basal transcription apparatus that assembles near the start site and is sufficient to initiate minimal levels of transcription. Transcriptional activator proteins, which bind to specific DNA sequences and bring about higher levels of transcription.

Promoters Core Promoter: located immediately upstream of the gene and has one or more consensus sequences. The most common of these consensus sequences is the TATA box, which has the consensus sequence TATAAA and is located from 25 to 30 bp upstream of the start site. TFIIB recognition element (BRE), which has the consensus sequence G/C G/C G/C C G C C and is located from 32 to 38 bp upstream of the start site. Instead of a TATA box, some core promoters have an initiator element (Inr) that directly overlaps the start site and has the consensus Y Y A N T/A Y Y. Downstream core promoter element (DPE) is found approximately 30 bp downstream of the start site in many promoters that also have Inr; the consensus sequence of DPE is R G A/T C G T G.

Promoters Core promoter are recognized by transcription factors that bind to them and serve as a platform for the assembly of the basal transcription apparatus

Promoters Regulatory Promoter: Transcriptional activator proteins bind to these sequences, affect the rate at which transcription is initiated Some regulatory promoters also contain repressing sequences, which are bound by proteins that lower the rate of transcription

Assembly of basal transcription apparatus

Assembly of basal transcription apparatus TFIID binds to the TATA box and positions the active site of RNA polymerase II TFIID consists of at least nine polypeptides. One of them is the TATA-binding protein (TBP) TFIIA helps to stabilize the interaction between TBP and DNA TFIIB plays a role in the selection of the start site TFIIH has helicase activity and unwinds the DNA during transcription The mediator plays a role in communication between the basal transcription apparatus and transcriptional activator proteins

Enhancers DNA sequences that increase the rate of transcription at distant genes are called enhancers Enhancers can stimulate any promoter in their vicinities Enhancer may be upstream or downstream from the affected gene or, in some cases, within an intron of the gene itself Enhancers also contain sequences that are recognized by transcriptional activator proteins Enhancers like sequences sometimes take part in repressing transcription called silencers.

Termination RNA polymerase I requires a termination factor, like the rho factor utilized in termination of some bacterial genes. 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 ends transcription after transcribing a terminator sequence that produces a string of Us 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 Us. 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.

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