Transcription bubbles

Figure 37-2. RNA polymerase (RNAP) catalyzes the polymerization of ribonucleotides into an RNA sequence that is complementary to the template strand of the gene. The RNA transcript has the same polarity (5 to 3 ) as the coding strand but contains L) rather than T. E coli RNAP consists of a core complex of two a subunits and two p subunits (P and p ). The holoen-zyme contains the 0 subunit bound to the ajPP core assembly. The co subunit is not shown. The transcription "bubble" is an approximately 20-bp area of melted DNA, and the entire complex covers 30-75 bp, depending on the conformation of RNAP.

F ig U re 1 3.1 A schematic view of RNA chain elongation catalyzed by an RNApolymerase. In the region being transcribed, the DNA double helix is unwound by about a turn to permit the DNAs sense strand to form a short segment of DNA-RNA hybrid double helix. That forms the transcription bubble. Note that the DNA bases in the bubble on the antisense strand are now exposed to the enzyme and are useable as a template for chain elongation. The RNApolymerase works its way down the DNA molecule until it encounters a stop signal. (Reproduced from D. Voet and J. G. Voet, Biochemistry, 3rd, edn, 2004 Donald and Judith G Voet. Reprinted with permission of John Wiley and Sons, Inc.)... 

Figure 11-3. The prokaryotic RNA transcription bubble. RNA pol II, RNA polymerase II hnRNA, heterogeneous nuclear RNA.

MECHANISM FIGURE 26-1 Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound, (a) About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA as shown facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. Red arrows show the direction in which the DNA must rotate to permit this process. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation. 

To enable RNA polymerase to synthesize an RNA strand complementary to one of the DNA strands, the DNA duplex must unwind over a short distance, forming a transcription bubble. During transcription, the E. coli RNA polymerase generally keeps about 17 bp unwound. The 8 bp RNA-DNA hybrid occurs in this unwound region. Elongation of a transcript by E. coli RNA polymerase proceeds at a rate of 50 to 90 nucleotides/s. Because DNA is a helix, movement of a transcription bubble requires considerable strand rotation of the nucleic acid molecules. DNA strand rotation is restricted... 

RNA Polymerase (a) How long would it take for the E. coli RNA polymerase to synthesize the primary transcript for the E. coli genes encoding the enzymes for lactose metabolism (the 5,300 bp lac operon, considered in Chapter 28) (b) How far along the DNA would the transcription bubble formed by RNA polymerase move in 10 seconds ... 

Figure 28-4 (A) Hypothetical structure of a "transcription bubble" formed by an RNA polymerase. Shown is a double-stranded length of DNA with the unwound bubble in the center. This contains a short DNA-RNA hybrid helix formed by the growing mRNA. The DNA double helix is undergoing separation at point A as is the hybrid helix at point B. NTP is the ribonucleotide triphosphate substrate. See Yager and von Hippel.71 (B) Stereoscopic view of the structure of RNA polymerase from Thermus aquaticus in a complex with a promoter DNA. Included are the al, all, (0, (3, P, and a subunits. However, the a C-terminal domains have been omitted. The template (t) strand passes through a tunnel, which is formed by the P and P subunits and two of the structural domains of the a subunit. The nontemplate (nt) strand follows a different path. The position of the -10, -35, and UP elements of the DNA are marked. From Murakami et al.33d Courtesy of Seth A. Darst.

Initiation of an RNA chain begins by reaction within the transcription bubble of either ATP or GTP with a second ribonucleotide triphosphate (Eq. 28-2) to form a dinucleotide still bearing a triphosphate at the 5 end. Further addition of nucleotide units at the 3 end by the same type... 

Rho and other termination factors. Termination proteins can also react with specific regions of DNA or of an RNA transcript to terminate transcription.183 The best known termination factor is the rho protein a hexamer of 45-kDa subunits. It interacts with transcripts at specific termination sequences, which are often C-rich, and in a process accompanied by hydrolysis of ATP causes release of both RNA and the polymerase from the DNA.192193 Additional E. coli proteins, products of genes nus A and nus G, cooperate with the rho factor at some termination sequences.194-196c The rho hexamer is a helicase that moves along the RNA transcript in the 5 —> 3 direction driven by ATP hydrolysis. If it locates an appropriate termination signal, it may utilize its helicase activity to uncoil the DNA-RNA hybrid segment within the transcription bubble (Fig. 28-4).197 198b... 

Following initiation, the a subunit dissociates from RNA polymerase to leave the core enzyme ( 2PP m) that continues RNA synthesis in a 5 — 3 direction using the four ribonucleoside 5 triphosphates as precursors. The DNA double helix is unwound for transcription, forming a transcription bubble, and is then rewound after the transcription complex has passed. 

Fig. 3. A transcription bubble. The DNA double helix Is unwound and RNA polymerase then synthesizes an RNA copy of the DNA template strand. The nascent RNA transiently forms an RNA-DNA hybrid helix but then peels away from the DNA which Is subsequently rewound into a helix once more.

The second type of terminator lacks the U region and requires the p termination factor for RNA chain release. The p factor is a hexamer of 45 kD subunits that binds a stretch of 72 nucleotides of single-stranded RNA. The p factor hydrolyzes ribonucleoside triphosphates to nucleoside diphosphates in the presence of single-stranded RNA and moves unidirectionally along nascent mRNA toward the transcription bubble and breaks the RNA-DNA hybrid, pulling away the RNA. 

The bacterial RNA polymerase has a subunit composition of ap>3 a, the a subunit being involved in correct initiation of transcription. Appropriate regulatory protein binding to the promoter region permits correct RNA polymerase binding, double-stranded DNA (dsDNA) unwinding and correct initiation of transcription. The dsDNA unwinds and the nascent RNA forms a transient RNA-DNA hybrid in the transcription bubble of unwound DNA that moves down the DNA. 

Elongation Takes Place at Transcription Bubbles That Move Along the DNA Template... 

How does this combination hairpin-oligo(U) structure terminate transcription First, it seems likely that RNA polymerase pauses immediately after it has synthesized a stretch of RNA that folds into a hairpin. Furthermore, the RNA-DNA hybrid helix produced after the hairpin is unstable because its rU-dA base pairs are the weakest of the four kinds. Hence, the pause in transcription caused by the hairpin permits the weakly bound nascent RNA to dissociate from the DNA template and then from the enzyme. The solitary DNA template strand rejoins its partner to re-form the DNA duplex, and the transcription bubble closes. 

Figure 28.8. Transcription Bubble. A schematic representation of a transcription bubble in the elongation of an RNA transcript. Duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end. The RNA-DNA hybrid rotates during elongation. 

Between bubbles. How far apart are transcription bubbles on E. coli genes that are being transcribed at a maximal rate ... 

Hence, the pause in transcription caused by the hairpin permits the weakly bound trascent RNA to dissociate frum the DNA template and then from the enzyme. The solitary DNA template strand rejoins its partner to re-form the DNA duplex, and the transcription bubble closes. 

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