Section K—Transcription in prokaryotes K1 Basic principles of transcription K2 Escherichia coli RNA polymerase K3 The E.coli σ70 promoter K4 Transcription initiation, elongation and termination

K1: Basic Principles of Transcription in Prokaryotes Transcription: A process catalyzed by RNA polymerase which DNA acts as a template for the synthesis of RNA. Transcription is the first and a vital control point in the expression of many genes. Here, we will focus on the basic mechanism of transcription. We will examine RNA polymerase, the enzyme that catalyzes transcription. The process begins when an RNA polymerase docks at a promoter, continues as the polymerase elongates the RNA chain, and ends when the polymerase reaches a terminator, and finally releases the transcript.

Central Dogma of molecular biology Information store in DNA flows: Overview RNA is a versatile molecule. In its most familiar role, RNA acts as an intermediary, carrying genetic information from the DNA to the machinery of protein synthesis. RNA also plays more active roles, performing many of the catalytic and recognition functions normally reserved for proteins. In fact, most of the RNA in cells is found in ribosomes--our protein-synthesizing machines--and the transfer RNA molecules used to add each new amino acid to growing proteins. In addition, countless small RNA molecules are involved in regulating, processing and disposing of the constant traffic of messenger RNA. RNA synthesis Similar to DNA synthesis Only one DNA strand serves as template Four types of RNA made Messenger RNA Transfer RNA Ribosomal RNA Small Nuclear RNA (eukaryotes) Central Dogma of molecular biology Information store in DNA flows: From DNA→DNA (Replication) From DNA →RNA (Transcription) From RNA →Protein (Translation)

RNA Synthesis RNA is transcribed 5’ →3’ like DNA DNA template strand is 3’ →5’ DNA non-template strand is 5’ →3’ RNA polymerase catalyzes addition of ribonucleotide triphosphates by phosphodiester bonds No primer required

Copyright 2000 John Wiley and Sons, Inc. nonsense sense RNA polymerase ATP GTP CTP UTP As you might expect, transcription follows the same base-pairing rules as DNA-replication: T, G, C and A in the DNA pair with A, C, G, and U, respectively in the RNA product. (Notice that uracil appears in RNA in place of thymine in DNA.) To some extent, this base-pairing pattern ensures that an RNA transcript is a faithful copy of the gene. Copyright 2000 John Wiley and Sons, Inc.

3’ 5’ DNA template strand C G Phosphodiester bond formation G C U A After the first nucleotide is in place, the polymerase joins a second nucleotide to the first, forming the initial phosphodiester bond in the RNA chian. In the same way, nucleotide joins the growing RNA chain at each step until transcription is complete. G Growing RNA 5’ strand T 5’

Steps in Transcription-Initiation，Elongation, and Termination Binding of RNA polymerase to the promoter sequence.-Promoters are sequences of DNA at the start of genes. Localized unwinding of the two strands of DNA.-In order to allow the template strand to be used for base pairing. Addition of first few rNTPs in the mRNA.-The polymerase then initiates the synthesis of the RNA strand at a specific nucleotide called start site. The RNA polymerase and its co-factors, when assembled on the DNA template, are often referred to as the transcription complex.

Copyright 2000 John Wiley and Sons, Inc. Promoter-These RNA polymerase binding sites are called promoters. Copyright 2000 John Wiley and Sons, Inc.

Elongation 2. Chain Elongation - Takes place within the transcription bubble - Adds complimentary rNTPs to the template DNA strand 5’ to 3’ to build mRNA transcript - Unwinds DNA ahead of the polymerization site - Rewinds the complimentary DNA strands behind the polymerization site - Continues this process until reaches termination sequence in DNA

Termination 3. Termination rho-independent termination: when mRNA bases bind to DNA of termination sequence (GC-rich region), stem-loop or hairpin form which inhibit further movement of RNA polymerase -mRNA transcript is released rho-dependent termination: when RNA polymerase reaches termination DNA sequence, rho protein is activated - rho binds to RNA polymerase altering its DNA binding site - RNA polymerase is released from template DNA strand, freeing mRNA transcript

RNA hairpin structure If we always read the strand that runs 5’-3’ left to right. The transcript of this sequence is self complementary around a center, which can form a loop. That means that the self-complementary bases can pair to form a hairpin as shown in this picture.

K2: E.coli RNA polymerase RNA polymerase is a huge factory with many moving parts. It is composed of several different proteins. Together, they form a machine that surrounds DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches confidently along the DNA copying RNA strands thousands of nucleotides long. Of course, highly directed chemical reactions such as transcription do not happen at significant rates by themselves-they are enzyme-catalyzed. The enzyme that directs transcription is call RNA polymerase.

Kornbergs with the polymerases Arthur Kornberg (left) with his son, Roger, after Roger received the Nobel Prize in Chemistry for 2006. Arthur Kornberg received the Nobel Prize in Physiology or Medicine in 1959. Both father and son are faculty members at the Stanford University School of Medicine. "There have got to be tens of thousands of people around the world today whose eyes are tearing up with the news that he's gone." "He was an extraordinary scientist. His accomplishments might be called legendary."

Subunits of E.coli RNA polymerase The complete enzyme, consisting of the core enzyme plus the σfactor, is called the holoenzyme. ω Cartoon illustrating separation of the subunits of E. coli RNA polymerase by SDS-PAGE

Roles of subunits Multi-subunit protein complex α2 ββ’σω α2 →Core protein assemble, promoter recognition β →Chain initiation, elongation β’ → DNA binding σ → Promoter recognition and binding ω → Unknown

Key points about the RNA polymerase The catalytic agent in the transcription process is RNA polymerase. The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a σ-factor, which directs the core to transcribe specific genes. The σ-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter. What’s the difference between the core and the holoenzyme? Which factor can stimulate initiation of transcription?

Copyright 2000 John Wiley and Sons, Inc. K3: σ 70 promoter of E.coli Promoter-These RNA polymerase binding sites are called promoters. Copyright 2000 John Wiley and Sons, Inc.

Strong/weak promoter Prokaryotic promoters contain two regions centered at -10 and -35 upstream of the transcription start site. In E. coli, these have the consensus sequences TATAAT and TTGACA, respectively. In general, the more closely regions within a promoter resemble these consensus sequences, the stronger that promoter will be. Some extraordinarily strong promoters contain an extra element ( an UP element) upstream of the core promoter. This makes these promoters better binding sites for RNA polymerase.

Transcription start site (TSS) TTGACA---------16-18bp--------TATAAT--------5-8bp-------C－T G A -35 sequence -10 sequence +1 The transcription start site is a purine in 90% of all genes. G is more common at the transcription start site than A. often, there are C and T bases on either side of the strat site nucleotide. (See above).

Promoter efficiency the -35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase σfactor; the -10 region is important for DNA unwinding; the sequence around the start site influences initiation; the sequence of the first 30 bases to be transcribed also influences transcription.

RNA synthesis (Transcription occurs in 3 stages) K4：Initiation, elongation and termination of transcription RNA synthesis (Transcription occurs in 3 stages) Initiation Elongation Termination 起始 延伸 终止

Initiation The initiation of transcription represented (1) formation of a closed promoter complex; (2) conversion of the closed promoter complex; (3) polymerizing the first few nucleotides while the polymerase remains at the promoter; and (4) promoter clearance, in which the transcript becomes long enough to form a stable hybrid with the template strand.

5’ to 3’-direction of chain growth U A T DNA template strand Growing RNA 5’ strand 5’ 3’ 5’ to 3’-direction of chain growth Elongation During the elongation phase of transcription, RNA polymerase directs the sequential binding of ribonucleotides to the growing RNA chain in the 5’-3’ direction (from the 5’-end toward the 3’-end of the RNA). As it does so, it moves along the DNA template, and the bubble of melted DNA moves with it.

Transcription bubble moves with the polymerase RNA polymerase This points to two fundamental differences between transcription and DNA replication: (a) RNA polymerase makes only one RNA strand during transcription, which means that it copies only one DNA strand in a given gene. (However, the opposite strand may be transcribed in another gene.). (b) In transcription, DNA melting is limited and transient. Only enough strand separation occurs to allow the polymerase to “read” the DNA template strand. However, during replication, the two parental DNA strands separate permanently. DNA template strand RNA Locally unwound segment of DNA On binding to a promoter, RNA polymerase causes the melting of at least 12bp, but probably about 17bp in the vicinity of the transcription start site. This transcription bubble moves with the polymerase, exposing the template strand so it can be transcribed.

Chain elongation The polymerase moves in a straight line, as indicated by the yellow arrow. This avoids twisting the RNA product around the DNA, but it forces the DNA ahead of the moving polymerase to untwist and the DNA behind the polymerase to twist back up again.

Termination Termination－When the polymerase reaches a terminator at the end of a gene it falls off the template, releasing the RNA. E. coli cells contain about equal numbers of two kinds of terminators. The first kind, known as intrinsic terminators, function with the RNA polymerase by itself WITHOUT help from proteins. The second kind depend on an auxiliary factor called rho (ρ). Naturally, these are called rho-dependent terminators.

The model of “hairpin”/inverted repeats

rho-independent termination First, The polymerase has paused at a string of weak rU-dA base pairs, and a hairpin has started to form just upstream of these base pairs.Then, as the hairpin forms, it further destabilized the RNA-DNA hybrid. This destabilization could take several forms: the formation of the hairpin could physically pull the RNA out of the polymerase; or it could simply cause the transcription bubble to collapse, expelling the RNA from the hybrid. At the end, the RNA product and polymerase dissociate completely from the DNA template, terminating transcription.

rho-dependent termination As polymerase makes RNA, rho binds to the transcript at a rho loading site and pursues the polymerase. When the hairpin forms in the transcript, the polymerase pauses, giving rho a chance to catch up. Finally, rho helicase unwinds the RNA-DNA hybrid and releases the transcript.

Error rate of transcription 1 mistake in 10,000 nucleotides incorporated into mRNA transcript No proofreading -RNA polymerase does not have 3’ to 5’ exonuclease activity so mistakes cannot be fixed -mutant mRNAs just never translate into functional proteins (mRNA with 5 min life span)

Homework What’re the main differences between the DNA replication and RNA transcription?