Transcription NEW

TRANSCRIPTION

SIDRA

Cells contain three major types of RNA: ribosomal RNA (rRNA), which constitutes two-thirds of the ribosomal mass;

transfer RNA (tRNA), a set of small, compact molecules that deliver amino acids to the ribosomes for assembly

into proteins; and messenger RNA (mRNA), whose nucleotide sequencesdirect protein synthesis. In addition, a host of other

noncoding RNA species play various roles in the regulation of gene expression and the processing of

newly transcribed RNA molecules

• RNA Polymerase Resembles Other Polymerases

• The E. coli RNAP holoenzyme is an 449-kD protein with subunit composition α2ββ’ωσ

• Once RNA synthesis has been initiated, however, the α subunit (also called the α factor) dissociates from the core enzyme α2ββ’ω,

• which carries out the actual polymerization process

• The DNA strand that serves as a template during transcription is known as the antisense or noncoding strand since its sequence is complementary to that of the RNA. The other DNA strand, which has the same sequence as the transcribed RNA (except for the replacement of U with T), is known as the sense or coding strand

Transcription Is Initiated at a Promoter

• Promoters consist of 40-bp sequences that are located on the 5’ side of the transcription start site.

• base pair in a promoter region is assigned a negative or positive number that indicates its position, upstream or downstream in the direction of RNAP travel, from the first nucleotide that is transcribed to RNA; this start site is 1 and there is no 0. Because RNA is synthesized in the 5¿ S 3¿ direction (see below), the promoter is said to lie upstream of the RNA’s starting nucleotide.

• Their most conserved sequence is a hexamerthe -10 position Pribnow box TATAAT

• Upstream sequences around position 35 also have a region of sequence similarity, TTGACA. The initiating (1) nucleotide, which is nearly always A or G, is centered in a poorly conserved CAT or CGT sequence

• rate of transcription is 20 to 50 nt/s at 37°C (but still many times slower than the DNA replication rate of 1000 nt/s; Section 25-2C). The error frequency in RNA synthesis is one wrong base incorporated for every 104 transcribed. This frequency, which is 104 to 106 times higher than that for DNA synthesis, is tolerable because most genes are repeatedly transcribed, because

• Many enzymes, particularly those involved in basic cellular “housekeeping” functions, are synthesized at a more or less constant rate; they are called constitutive enzymes

• Other enzymes, termed inducible enzymes, are synthesized at rates that vary with the cell’s circumstances.

Transcription Terminates at Specific Sites

• The transcription termination sequences of about half of E. coli genes share two common features (Fig. 26-10):

• 1. A series of 4 to 10 consecutive A T base pairs, with the A’s on the template strand. The transcribed RNA is terminated in or just past this sequence.

• 2. A G C–rich region with a palindromic sequence that immediately precedes the series of A T’s.

• they require the action of a protein known as Rho factor to terminate transcription

Eukaryotes Have Several RNA Polymerases

• RNA polymerase I (RNAP I), which is located in the nucleoli where ribosomes are assembled, synthesizes the precursors of most rRNAs

• RNA polymerase II (RNAP II), which occurs in the nucleoplasm, synthesizes the mRNA precursors.

• 3. RNA polymerase III (RNAP III), which also occurs in the nucleoplasm, synthesizes the precursors of 5S rRNA, the tRNAs, and a variety of other small nuclear and cytosolic RNAs

• RNAP I requires a so-called core promoter element, which spans positions -31 to +6 and hence overlaps the transcribed region. However, efficient transcription also requires an upstream promoter element, which is located between residues -187 and -107.

• TATA box, an AT rich sequence located 25 to 31 bp upstream from the transcription start site. The TATA box (consensus sequence TATAA/TAA/T resembles the -10 region of a prokaryotic promoter (TATAAT), although it differs in its location relative to the transcription start site (-27 versus -10).

• For instance, many eukaryotic structural genes have a conserved consensus sequence of CCAAT (the CCAAT box) located between about -70 and -90 whose alteration greatly reduces the gene’s transcription rate.

• The promoters of some genes transcribed by RNAP III are located entirely within the genes’ transcribed regions

• between nucleotides +40 and +80.

• Eukaryotic RNAPs, have molecular masses of as much as 600 kD, .Each eukaryotic RNAP contains two nonidentical “large” (>120 kD) subunits, which are homologs of the prokaryotic β and β’ subunits, and an array of up to 12 different “small” (<50 kD) subunits, two of which are homologs of the prokaryotic α subunit and one of which is a homolog of the ω subunit. Five of the small subunits, including the ω homolog, are identical in all three eukaryotic enzymes, and the αhomologs are identical in RNAPs I and III.

• RNAP II binds two Mg2 ions at its active site in the vicinity of five conserved acidic residues, which suggests that RNAPs catalyze RNA elongation via a two-metal ion mechanism similar to that employed by DNA polymerases

• the surface of RNAP II is almost entirely negatively charged except for the DNA-binding cleft and the region about the active site, which are positively charged.

• RNAP II’s contain Rpb1 subunit the homolog of the β’ subunit in prokaryotic RNAPs, has an extraordinary C-terminal domain (CTD). In mammals, the CTD contains 52 highly conserved repeats with the consensus sequence Pro-Thr-Ser-Pro-Ser-Tyr-Ser (26 repeats in yeast.

• 50 Ser residues in this hydroxylrich protein segment are subject to reversible phosphorylation by CTD kinases and CTD phosphatases.

general transcription factors (GTFs

• Protein factors bind selectively to the promoter regions of DNA. With class II promoters (those transcribed by RNAP II), a complex of at least six general transcription factors operates as a formal equivalent of a prokaryotic factor.

• TF (for transcription factor)

• preinitiation complex (PIC).

PIC Formation Often Begins with TATA-Binding Protein Binding to the

TATA Box• The first transcription factor to bind to TATA box–

containing promoters is the TATA-binding protein (TBP), which as its name indicates, binds to the TATA box and thereby helps identify the transcription start site. TBP is subsequently joined on the promoter by additional subunits to form, in humans, the ~1122-kD, 17-subunit complex TFIID.

• The highly conserved C-terminal domain of TBP contains two ~40% identical direct repeats of 66 residues separated by a highly basic segment

• The TBP, which undergoes little conformational change on binding DNA, does so via hydrogen bonding and van der Waals interactions. The kinked and partially unwound DNA is stabilized by a wedge of two Phe side chains on each side of the saddle structure that pry apart the two base pairs flanking each kink from their minor groove sides. The bent conformation of DNA creates a stage for the assembly of other proteins to form the PIC.

TFIIA, TFIIB, and TAFs Interact with TBP and RNAP II

• The PIC requires, at a minimum, TBP, TFIIB, TFIIE, TFIIF, and TFIIH. TFIIB consists of two domains, an N-terminal domain (TFIIBN), which interacts with RNAP II, and a C-terminal domain (TFIIBC), which binds DNA and interacts with TBP.

• Initiator element YYA+1NA/TYY

• The three proteins bind to the DNA just upstream from the transcription start site, leaving ample room for additional proteins and RNAP II to bind. Since the pseudosymmetric TBP has been shown to bind to the TATA box in either orientation, it appears that base-specific interactions between TFIIB and the promoter function to position TFIIB to properly orient the TBP on the promoter.

• The remaining components of TFIID, which are known as TBP-associated factors (TAFs), form a horseshoe-shaped complex to which TFIIA and TFIIB are bound.

• In the final steps of PIC formation (Fig. 26-17), TFIIF recruits RNAP II to the promoter in a manner reminiscent of the way that σ factor interacts with bacterial RNAP. In fact, the second largest of TFIIF’s three subunits is homologous to σ 70, the predominant bacterial σ factor, and, moreover, can specifically interact with bacterial RNAPs (although it does not participate in promoter recognition). Finally, TFIIE and TFIIH join the assembly. Once this complex has been assembled, the ATP-dependent helicase activity of TFIIH induces the formation of the open complex so that RNA synthesis can commence.

Promoters That Lack a TATA Box Also Bind TBP

• Since the TATA-binding protein is a component of TFIID, a general transcription factor for RNAP II. In many cases, the presence of the Inr element is sufficient to direct RNAP II to the correct start site. These systems require the participation of many of the same GTFs that initiate transcription from TATA box–containing promoters. Surprisingly, they also require TBP. This suggests that with TATA-less promoters, Inr recruits TFIID such that its component TBP binds to the -30 region in a sequence-nonspecific manner.

• TBP Is a Universal Transcription Factor

Elongation Requires Different Transcription Factors

• After RNAP II initiates RNA synthesis and successfully produces a short transcript, the transcription machinery undergoes a transition to the elongation mode. The switch appears to involve displacement of the finger domain of TFIIB, which would otherwise clash with the growing RNA chain in the active site, as well as phosphorylation of the C-terminal domain (CTD) of RNAP II’s Rpb1 subunit

• Phosphorylated RNAP II releases some of the transcription-initiating factors and advances beyond the promoter region. In fact, when RNAP II moves away from (“clears”) the promoter, it leaves behind some GTFs, including TFIID. These proteins can reinitiate transcription by recruiting another RNAP II to the promoter. Consequently, the first RNAP to transcribe a gene may act as a “pioneer” polymerase that helps pave the way for additional rounds of transcription

• During elongation, a six-protein complex called Elongator binds to the phosphorylatedCTD of Rpb1, taking the place of the jettisoned transcription factors. Although Elongator is not essential for transcription by RNAP II in vitro, its presence accelerates transcription. Interestingly, TFIIF and TFIIH remain associated with the polymerase during elongation

Eukaryotes Lack Precise Transcription Termination Sites.

• The sequences signaling transcriptional termination in eukaryotes have not been identified.

• This is largely because the termination process is imprecise; that is, the primary transcripts of a given structural gene have heterogeneous 3’ sequences.

• However, a precise termination site is not required because the transcript undergoes processing that includes endonucleolytic cleavage at a specific site