29.1 RNA Polymerases Catalyze Transcription

All cellular RNA molecules are synthesized by RNA polymerases according to instructions given by DNA templates. The activated monomer substrates are ribonucleoside triphosphates. The direction of RNA synthesis is 5′ → 3′, as in DNA synthesis. RNA polymerases, unlike DNA polymerases, do not need a primer.

RNA polymerase in E. coli is a multisubunit enzyme. The subunit composition of the ~500-kDa holoenzyme is α₂ββ′ ωσ and that of the core enzyme is α₂ββ′ ω. Transcription is initiated at promoter sites consisting of two sequences, one centered near −10 and the other near −35; that is, 10 and 35 nucleotides away from the start site in the 5′ (upstream) direction. The consensus sequence of the −10 region is TATAAT. The σ subunit enables the holoenzyme to recognize promoter sites. When the growth temperature is raised, E. coli expresses a special σ subunit that selectively binds the distinctive promoter of heat-shock genes. RNA polymerase must unwind the template double helix for transcription to take place. Unwinding exposes some 17 bases on the template strand and sets the stage for the formation of the first phosphodiester bond. Newly synthesized RNA chains usually start with pppG or pppA. The σ subunit usually dissociates from the holoenzyme after the initiation of the new chain.

Elongation takes place at transcription bubbles that move along the DNA template at a rate of about 50 nucleotides per second. RNA polymerase occasionally backtracks, a process that can facilitate proofreading of the RNA transcript. The nascent RNA chain contains stop signals that end transcription. One stop signal is an RNA hairpin, which is followed by several U residues. A different stop signal is read by the rho protein, an ATPase. Some genes are regulated by riboswitches, structures that form in RNA transcripts and bind specific metabolites. In E. coli, precursors of transfer RNA and ribosomal RNA are cleaved and chemically modified after transcription, whereas messenger RNA is used unchanged as a template for protein synthesis.

29.2 Transcription in Eukaryotes Is Highly Regulated

RNA synthesis in eukaryotes takes place in the nucleus, whereas protein synthesis takes place in the cytoplasm. There are three types of RNA polymerase in the nucleus: RNA polymerase I makes ribosomal RNA precursors, II makes messenger RNA precursors, and III makes transfer RNA precursors. Eukaryotic promoters are complex, being composed of several different elements. Promoters for RNA polymerase II may be located on the 5′ side or the 3′ side of the start site for transcription. A common type of eukaryotic promoter consists of a TATA box centered between −30 and −100 and paired with an initiator element. Eukaryotic promoter elements are recognized by proteins called transcription factors rather than by RNA polymerase II. The saddle-shaped TATA-box-binding protein unwinds and sharply bends DNA at TATA-box sequences and serves as a focal point for the assembly of transcription complexes. The TATA-box-binding protein initiates the assembly of the active transcription complex. The activity of many promoters is greatly increased by enhancer sequences that have no promoter activity of their own. Enhancer sequences can act over distances of several kilobases, and they can be located either upstream or downstream of a gene.

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29.3 The Transcription Products of Eukaryotic Polymerases Are Processed

The 5′ ends of mRNA precursors become capped and methylated in the course of transcription. A 3′ poly(A) tail is added to most mRNA precursors after the nascent chain has been cleaved by an endonuclease. RNA editing alters the nucleotide sequence of some mRNAs, such as the one for apolipoprotein B.

The splicing of mRNA precursors is carried out by spliceosomes, which consist of small nuclear ribonucleoprotein particles. Splice sites in mRNA precursors are specified by sequences at ends of introns and by branch sites near their 3′ ends. The 2′-OH group of an adenosine residue in the branch site attacks the 5′ splice site to form a lariat intermediate. The newly generated 3′-OH terminus of the upstream exon then attacks the 3′ splice site to become joined to the downstream exon. Splicing thus consists of two transesterification reactions, with the number of phosphodiester bonds remaining constant during reactions. Small nuclear RNAs in spliceosomes catalyze the splicing of mRNA precursors. In particular, U2 and U6 snRNAs form the active centers of spliceosomes.

The events in posttranscriptional processing of mRNA are controlled by the phosphorylation state of the carboxy-terminal domain, part of RNA polymerase II.

29.4 The Discovery of Catalytic RNA Was Revealing in Regard to Both Mechanism and Evolution

Some RNA molecules, such as those containing the group I intron, undergo self-splicing in the absence of protein. A self-modified version of this rRNA intron displays true catalytic activity and is thus a ribozyme. Spliceosome-catalyzed splicing may have evolved from self-splicing. The discovery of catalytic RNA has opened new vistas in our exploration of early stages of molecular evolution and the origins of life.