Mechanisms of Transcription 生物学基地班 魏昌勇

OUTLINE How the series of bases in the DNA directs the production of the RNAs and proteins that perform cellular functions and define cellular identity.

Objectives  Understand the structure of RNA polymerases  Understand the phases of the transcription cycle  Understand the differences between transcription and replication

Gene expression is the process by which the information in the DNA double helix is converted into the RNAs and proteins whose activities bestow upon a cell its morphology and functions. Transcription is the first step in the gene expression and involved copying DNA into RNA.

Transcription is, chemically and enzymatically, very similar to DNA replication, but there are some important differences:  RNA is made of ribonucleotides (rather than deoxyribonuleotides)  RNA polymerase catalyzes the reaction (does not need a primer)  The synthesized RNA does not remain base-paired to the template DNA strand  Less accurate (one in 10000,compared to one in for replication)  Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (replication must copy the entire genome and do so once every cell division)

Fig 12-1 Transcription of the DNA into RNA.(in the absence of the enzymes involved)

Multiple RNA polymerases can transcribe the same gene at the same time. A cell can synthesize a large number of RNA transcription in a short time.

Topic 1: RNA Polymerase and The Transcription Cycle

RNA Polymerases Comes in Different Forms, but Share Many Features. 1.RNA polymerases performs essentially the same reaction in the cell,from bacteria to humans. 2.The cellular RNA polymerases are made up of multiple subunits. 3.Bacteria have only a single RNA polymerases,while in eukaryotic cells there are three: RNA Pol Ⅰ，Ⅱ，Ⅲ. 4.Pol Ⅱ is the polymerases responsible for transcribing most genes——indeed, essentially all protein-encoding genes. 5.Pol Ⅰ and Pol Ⅲ are each involved in transcribing specialized, RNA-encoding genes.

Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases.

The shape of each enzyme resembles a crab claw.

Transcription by RNA polymerases Proceeds in a Series of steps  Initiation.  Elongation.  Termination.

Initiation  A promoter is the DNA sequence that initially binds the RNA polymerase.  Promoter-polymerase complex undergoes structural changes required for initiation to proceed.  The base at the transcription site unwinds and producing a “bubble” of single-stranded DNA.  Transcription always occurs in a 5’ to 3’ direction.

Transcription Initiation Involves three Defined Steps  The initial binding of polymerase to promoter to form what is called a closed complex.  The closed complex undergoes a transition to the open complex.  Promoter escape.

Promoter escape

Formation of a closed complex

Transition to an open complex

Elongation  Once the RNA polymerase has synthesized a short stretch of RNA(approximately ten bases),it shifts into the elongation phase.  This transcription requires further conformational change in polymerase that leads it to grip the template more firmly.  Function: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain from the template, performs proofreading.

Termination  Once the polymerase has transcribed the length of the gene (or genes), it must stop and released the RNA product. This step is called termination.  In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.

Topic 2: The Transcription Cycle in Bacteria

Bacteria Promoters Vary in Strength and Sequence, but Have Certain Defining Features The bacteria core RNA polymerases can, in principle, initiate transcription at any point on a DNA molecule. In cells, polymerases initiate transcription only at promoters. An initiation factor called σ that converts core enzyme into the form that initiates only at promoter. That form of the enzymes is called the RNA polymerase holoenzyme. In the case of E.coli, the predominant σ is called σ70. Promoter recognized by σ70 contains two conserved sequences (-35 and –10 regions/elements) separated by a non-specific stretch of nt. Position +1 is the transcription start site

Figure 12-5 Features of bacteria promoters. Various combination of bacteria promoter elements are shown. a. σ70 promoters contain recognizable –35 and –10 regions, but the sequences are not identical. b. UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase. c. Another class of s70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region.

The Factor Mediate Binding of Polymerase to the Promoter Fig 12-6 Regions of σ Those regions σ factor that recognize specific regions of the promoter are indicated by arrows. Region 2.3 is responsible for melting the DNA.

Figure 12-7 σ and α subunits recruit RNA polymerases core Enzyme to the promoter. The C-domain of the α subunit (α CTD) Recognize the UP-element, while α region 2 and 4 recognize the -10 and -35 regions respectively.

Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA Isomerization: essentially irreversible and, once complete, typically guarantees that transcription will subsequently initiate (though Regulation can still be imposed after this point in some cases.

Figure 12-8 Channels into and out of the open complex.

Transcription is Initiated by RNA Polymerase without the Need for a Primer

RNA Polymerase Synthesizes Several Short RNAs Before Entering the Elongation Phase

The Elongating Polymerase is a Processive Machine that Synthesizes and Proofreads RNA

Transcription is terminated by signals within the RNA sequence  Terminators: the sequence that trigger the elongating polymerases to dissociate the DNA and release the RNA chain it has made.  In bacteria, terminators come in two type: Rho- independent and Rho-dependent (also called intrinsic terminators).  Rho-dependent terminator need Rho protein.

Figure 12-9 Sequence of a Rho-dependent terminator. At the top is the sequence, in the DNA, of the terminator. Below is shown the sequence of the RNA, and at the bottom the structure of the terminator hairpin.

Figure Transcription termination. Shown is a model for how the Rho-independent terminator might work.

Figure The ρ transcription termination factor. The crystal structure of the Rho termination factor of shown in a top down view.

Topic 3 Transcription in Eukaryotes

RNA Polymerase II Core Promoters Are Made up of Combinations of Four Different Sequence Elements  The eukaryotic are core promoter refers to the minimal set of sequence element required for accurate transcription initiation by the Pol II machinery in vitro.  Beyond——and typically upstream of —— the core promoter elements required for efficient transcription in vivo. Together these element constitute the regulatory sequences.

Regulatory sequence: promoter proximal elements, upstream Activator sequences (UASs), enhancers, and a series of repressing elements called silencers, boundary elements, and insulators.

RNA Pol II Forms a Pre-Initiation Complex with GTFs at the Promoter Pre-initiation complex, TBP, TAFs. TATA sequence TF IID, TF IIA, TF IIB, TF IIF, TF IIE, TF IIH.

Figure Transcription initiation by RNA polymerase II. The step-wide assembly of the Pol II pre-initiation complex is shown here.

TBP Binds to and Distorts DNA Using a  Sheet Inserted into the Minor Groove Figure TBP-DNA complex. The TATA binding protein (TBP) is shown here in purple complexed with the DNA TATA sequence (shown in gray) found at the start of many Pol II genes.

The Other General Transcription Factors also Have Specific Roles in Initiation  TAFs. TBP is associated with about ten TAFs. Another TAF appears to regulate the binding of TBP to DNA.  TF IIB. This protein, a single polypeptide chain, enters the pre-initiation complex after TBF.  TF IIF.  TF IIE and TF IIH.

Figure TFIIB-TBP-promoter Complex. this structure shows the TBP protein bound to the TATA sequence.

In vivo, Transcription Initiation Requires Additional Proteins Figure Assembly of the pre- initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators.

Mediator Consists of Many Subunits, Some Conserved From Yeast to Human Figure12-17 Comparison of the yeast and human Mediators. The homologous are shown in blue.

A New Set of Factors Stimulate Pol II Elongation and RNA Proofreading Figure RNA processing enzymes are recruited by the tail polymerases.

Elongation Polymerase is Associated with a New Set of Protein Factors Required for Various Types of RNA Processing  Poly (A) tail.  5’cap.  Splicing.  3’ end polyadenylation

Figure The structural and formation of the 5’RNA cap. RNA processing a 5’ end capping.

Figure Polyadenylation and Termination.

RNA Pol I and III Recognize Distinct Promoters, Using Distinct Sets of Transcription Factors, but still Require TBP I is required for the expression of only one gene, that encoding the rRNA precursor.  Pol I is required for the expression of only one gene, that encoding the rRNA precursor.  Pol III promoters come in various forms, and the vast majority have the unusual features of being located downstream of the transcription start site.

I promoter region. Figure Pol I promoter region. a.Structure of the Pol I promoter. b.Pol I txn factors.

III core promoter. Figure Pol III core promoter. Shown here is the promoter for a yeast tRNA gene.

Summary RNA polymerase  is the primary enzyme of the transcription  Resembles a crab claw  is generally composed of several subunits  Add nucleotides to the 3’ end of the growing RNA chain

Transcription cycle  Initiation (Formation of a closed complex, Transition to a open complex, promoter escape)  Elongation (Unwind the DNA in front of the enzyme, Synthesis of the RNA,RNA proofreading, Dissociation of RNA, Re-annealing of the DNA behind the enzyme)  Termination (Rho-independent terminators, Rho- dependent terminators)