RNA Synthesis and Processing

Introduction

Regulation of gene expression allows cells to adapt to environmental changes and is responsible for the distinct activities of the differentiated cell types that make up complex organisms.

Transcription is the first step in gene expression, and the initial level at which gene expression is regulated.

RNAs in eukaryotic cells are then modified and processed in various ways.

Transcription in Prokaryotes

RNA polymerase catalyzes polymerization of ribonucleoside 5′-triphosphates (NTPs) as directed by a DNA template, always in the 5′ to 3′ direction.

Transcription initiates de novo (no preformed primer required) at specific sites—this is a major step at which regulation of transcription occurs.

Transcription in ProkaryotesBacterial RNA polymerase has five types of subunits.

The σ subunit is weakly bound and can be separated from the others. It is required to identify the correct sites for transcription initiation.

Most bacteria have several different σ’s that direct RNA polymerase to different start sites under different conditions.

Transcription in Prokaryotes

The promoter is the gene sequence to which RNA polymerase binds to initiate transcription.

Promoters have six nucleotides and are located at 10 and 35 base pairs upstream of the transcription start site.

Consensus sequences are the bases most frequently found in different promoters.

Transcription in ProkaryotesExperiments have shown the functional importance of the –10

and –35 promoter elements.

The sites at which RNA polymerase binds to promoters have been directly identified by DNA footprinting.

A DNA fragment is labeled at one end with a radioisotope or fluorescent dye.

The labeled DNA is incubated with RNA polymerase and then partially digested with DNase.

Regions of DNA where the protein binds are protected from DNase digestion, and can be identified by comparing to DNA with no bound protein.

Figure 7.3 DNA footprinting

Transcription in Prokaryotes

Footprinting analysis has shown that the σ subunit binds specifically to sequences in both the –35 and –10 promoter regions, substantiating the importance of these sequences in promoter function.

σ binds specifically to both the –35 and –10 sequences, leading to the initiation of transcription at the beginning of a gene.

Initial binding is referred to as a closed-promoter complex because the DNA is not unwound.

The polymerase then unwinds 12–14 bases of DNA to form an open-promoter complex, allowing transcription.

After addition of about ten nucleotides, σ is released from the polymerase.

The polymerase continues elongation of the RNA chain.

Figure 7.4 Transcription by E. coli RNA polymerase (Part 1)

Figure 7.4 Transcription by E. coli RNA polymerase (Part 2)

Transcription in ProkaryotesDuring elongation, polymerase maintains an unwound region of about 15 base pairs.

High-resolution structural analysis shows that the β and β′ subunits form a crab claw-like structure that grips the DNA template. An internal channel between these subunits contains the polymerase active site.

Transcription in Prokaryotes

RNA synthesis continues until the polymerase encounters a termination signal.

The most common signal is a symmetrical inverted repeat of a GC-rich sequence followed by seven A residues.

Transcription of the GC-rich inverted repeat results in a segment of RNA that can form a stable stemloop structure.

This disrupts its association with the DNA template and terminates transcription.

Figure 7.6 Transcription termination

Transcription in Prokaryotes

Alternatively, transcription of some genes is terminated by a specific termination protein (Rho), which binds extended segments of single-stranded RNA.

Most transcriptional regulation in bacteria operates at initiation.

Studies of gene regulation in the 1950s used enzymes involved in lactose metabolism.

The enzymes are only expressed when lactose is present.

Transcription in Prokaryotes

β-galactosidase cleaves lactose into glucose and galactose.

Lactose permease transports lactose into the cell.

Transacetylase is thought to inactivate toxic thiogalactosides that are transported into the cell along with lactose.

Transcription in ProkaryotesGenes encoding these enzymes are expressed as a single unit, called an operon.

Transcription of the operon is controlled by o (operator), adjacent to the transcription initiation site.

The i gene (not physically linked to the operon), encodes a protein that binds to the operator.

Transcription in Prokaryotes

Mutants that don’t produce i gene product express the operon even when lactose is not available.

This implies that the normal i gene product is a repressor, which blocks transcription when bound to o.

In normal cells, lactose binds to the repressor, preventing it from binding to the operator, and the genes are expressed.

The lactose operon illustrates the central principle of gene regulation: control of transcription is mediated by the interaction of regulatory proteins with specific DNA sequences.

Transcription in Prokaryotes

Cis-acting control elements only affect the expression of linked genes on the same DNA molecule (e.g. the operator).

Trans-acting factors can affect expression of genes located on other chromosomes (e.g. the repressor).

The lac operon is an example of negative control—binding of the repressor blocks transcription.

An example of positive control in E. coli :Presence of glucose (the preferred energy source)

represses expression of genes for enzymes that break down other sugars, such as the lac operon.

Transcription in Prokaryotes

Low glucose levels activate adenylyl cyclase, which converts ATP to cAMP.

cAMP then binds to catabolite activator protein (CAP).

CAP then binds to its target DNA sequences, 60 bases upstream of the transcription start site in the lac operon.

Eukaryotic RNA Polymerases and General Transcription FactorsEukaryotic cells have three nuclear RNA polymerases that transcribe different

classes of genes.

They are complex enzymes, consisting of 12 to 17 different subunits each.

They all have 9 conserved subunits, 5 of which are related to subunits of bacterial RNA polymerase.

Eukaryotic RNA Polymerases and General Transcription Factors

RNA polymerase II is responsible for synthesis of mRNA and it has been the focus of most transcription studies.

Unlike prokaryotic RNA polymerase, it requires initiation factors that (in contrast to bacterial σ factors) are not associated with the polymerase.

Eukaryotic RNA Polymerases and General Transcription Factors

General transcription factors are proteins involved in transcription from all polymerase II promoters.

About 10% of the genes in the human genome encode transcription factors, emphasizing the importance of these proteins.

Promoters contain several different regulatory sequence elements.

Promoters of different genes contain different combinations of promoter elements, which appear to function together to bind general transcription factors.

Figure 7.11 Formation of a polymerase II preinitiation complex in vitro (Part 1)

Eukaryotic RNA Polymerases and General Transcription Factors

Sequence elements include the TATA boxwhich resembles the –10 sequence element of bacterial promoters.

A minimum of five general transcription factors are required for initiation of transcription in vitro.

The first step is binding of general transcription factor TFIID, composed of multiple subunits, including the TATA-binding protein (TBP) and 14 other polypeptides, called TBP-associated factors (TAFs).

Eukaryotic RNA Polymerases and General Transcription Factors

Following recruitment of RNA polymerase II to the promoter, the binding of two additional factors (TFIIE and TFIIH) completes formation of the preinitiation complex.

Eukaryotic RNA Polymerases and General Transcription FactorsWithin a cell, additional factors are required to initiate transcription.

These include Mediator, a large protein complex of more than 20 subunits; it interacts with both general transcription factors and RNA polymerase.

Eukaryotic RNA Polymerases and General Transcription FactorsRNA polymerase I is devoted solely to transcription of rRNA genes, which

are present in tandem repeats.

Transcription yields a large 45S pre-rRNA, which is processed to yield the 28S, 18S, and 5.8S rRNAs.

Eukaryotic RNA Polymerases and General Transcription Factors

Promoters of rRNA genes are recognized by two transcription factors, UBF (upstream binding factor) and SL1 (selectivity factor 1), which recruit polymerase I.

SL1 transcription factor is composed of four subunits, one of which is TBP.

Eukaryotic RNA Polymerases and General Transcription FactorsGenes for tRNAs, 5S rRNA, and some of the small RNAs are transcribed by

polymerase III.

Promoters of 5S rRNA and tRNA genes are downstream of the transcription initiation site.

The promoter of the U6 snRNA gene is upstream of the transcription start site and contains a TATA box.

Regulation of Transcription in Eukaryotes

An important difference between transcriptional regulation in prokaryotes and eukaryotes results from the packaging of eukaryotic DNA into chromatin.

Modifications of chromatin structure play key roles in the control of transcription in eukaryotic cells.

Many cis-acting sequences regulate expression of eukaryotic genes.

Some eukaryotic regulatory sequences have been identified by gene transfer assays.

Regulation of Transcription in EukaryotesRegulatory sequences are ligated to a reporter gene that encodes an easily

detectable enzyme, such as firefly luciferase.

The regulatory sequence then directs expression of the reporter gene in cultured cells.

Two cis-acting regulatory sequences were identified by studies of the promoter of the herpes simplex virus gene that encodes thymidine kinase.

Regulation of Transcription in Eukaryotes

Some regulatory sequences are farther away—called enhancers.

They were first identified during studies of the promoter of another virus, SV40.

Regulation of Transcription in EukaryotesActivity of enhancers doesn’t depend on either their distance

from, or orientation with respect to, the transcription initiation site.

Figure 7.20 Action of enhancers (Part 2)

Regulation of Transcription in EukaryotesEnhancers, like promoters, function by binding transcription factors that then

regulate RNA polymerase.

DNA looping allows a transcription factor bound to a distant enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter.

Regulation of Transcription in EukaryotesExample: an enhancer controls transcription of immunoglobulin genes in B

lymphocytes.

Gene transfer experiments show that the enhancer is active in lymphocytes, but not in other types of cells.

This regulatory sequence is partly responsible for tissue-specificexpression of the immunoglobulin genes.

Enhancers usually contain multiple sequence elements that bind different transcriptional regulatory proteins.

The immunoglobulin heavy-chain enhancer spans about 200 base pairs and contains at least nine distinct sequence elements that serve as protein-binding sites.

Regulation of Transcription in Eukaryotes

The immunoglobulin enhancer contains negative regulatory elements that inhibit transcription in inappropriate cell types; and positive regulatory elements that activate transcription in B lymphocytes.

The overall activity reflects the combined action of the proteins associated with each of the sequence elements.

Activity of any given enhancer is specific for the promoter of its appropriate target gene.

This specificity is maintained in part by insulators or barrier elements, which divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain.

Regulation of Transcription in EukaryotesTranscription factor binding sites have been identified by DNA footprinting and

electrophoretic-mobility shift assay (EMSA).

Radiolabeled DNA fragments are incubated with a protein and then subjected to electrophoresis through a nondenaturing gel.

Migration of a DNA fragment through the gel is slowed by a bound protein.

Regulation of Transcription in EukaryotesBinding sites are usually short DNA sequences (6–10 base pairs) and they

are degenerate—the transcription factor will bind to the consensus sequence, but also to sequences that differ from the consensus at one or more positions.

Transcription factor binding sites are shown as pictograms, representing the frequency of each base at all positions of known binding sites for a given factor.

Regulation of Transcription in EukaryotesChromatin immunoprecipitation identifies DNA regions that bind to transcription

factors.

Cells are treated with formaldehyde to cross-link transcription factors to the DNA sequences to which they were bound.

Chromatin is extracted and fragmented. Fragments of DNA linked to a transcription factor can then be isolated by immunoprecipitation.

Regulation of Transcription in Eukaryotes

One of the first transcription factors to be isolated was Sp1, in studies of virus SV40 DNA, by Tjian and colleagues.

Sp1 binds to GC boxes in the SV40 promoter. This established the action of Sp1 and also suggested a method for purification of transcription factors.

Regulation of Transcription in EukaryotesDNA-affinity chromatography:

Double-stranded oligonucleotides with repeated GC box sequences are bound to agarose beads in a column.

Cell extracts are passed through the column. Sp1 binds to the GC box with high affinity and is retained on the column.

Regulation of Transcription in EukaryotesTranscriptional activators, like Sp1, bind to regulatory DNA sequences

and stimulate transcription.

These factors have two independent domains: one region binds DNA, the other stimulates transcription by interacting with other proteins, such as Mediator.

Regulation of Transcription in EukaryotesMany different transcription factors have now been identified in eukaryotic

cells.

About 2000 are encoded in the human genome.

They contain many distinct types of DNA-binding domains.

The most common is the zinc finger domain, which binds zinc ions and folds into loops (“fingers”) that bind DNA.

Steroid hormone receptors contain zinc fingers; they regulate gene transcription in response to hormones such as estrogen and testosterone.

Figure 7.28 Examples of DNA-binding domains (Part 2)

Helix-turn-helix domain: one helix makes most of the contacts with DNA, the other helices lie across the complex to stabilize the interaction.

They include homeodomain proteins, important in the regulation of gene expression during embryonic development.

Regulation of Transcription in EukaryotesHomeodomain proteins were first discovered as developmental mutants in

Drosophila.

They result in development of flies in which one body part is transformed into another.

In Antennapedia, legs rather than antennae grow from the head.1