GENERACIÓN DE MODELO DE DESEMPEÑO

1.3.5.1 Introduction

A number of mechanisms have evolved that prevent the termination of transcription. Transcription antitermination mechanisms regulating gene expression may be described as processive or non-processive (Greenblatt et al., 1993).

In processive antitermination the RNAP is modified such that it transcribes through downstream transcriptional terminator sequences. This is the case for the bacteriophage

X N protein. Processive antitermination by N is dependent on an RNA N utilization site

(nut site) onto which assembles a complex containing N and four E. coli proteins, NusA, NusB, NusG and ribosomal protein SIO (Greenblatt et al., 1993). This ribonucleoprotein complex associates stably with RNAP during elongation, converting it into a ‘juggernaut’ which transcribes through downstream terminators. A similar role

is exhibited by the HIV-1 Tat proteins which interact with numerous components of a transcription complex including RNAPII, during viral replication in the eukaryotic host (Mavankal et al.y 1996).

Non-processive antitermination involves a regulatory protein or ribosome binding to the mRNA at a specific site and preventing the formation of a terminator structure. Although some authors use the term attenuation to describe non-processive antitermination, attenuation is a term best reserved for the ribosome-dependent antitermination mechanisms exhibited by the biosynthetic opérons of a number of amino acids such as tryptophan, histidine, threonine and phenylalanine. The tryptophan (trp)

operon of E. coli will be used as an example to illustrate attenuation. In E. coli the biosynthesis of tryptophan is negatively regulated by the tryptophan bound repressor complex and positively regulated by attenuation (Landick et al., 1996). The leader region of the trp operon contains a promoter, a short ORF containing tryptophan codons followed by the first structural gene, all of which are transcribed on the same polycistronic mRNA. This polycistronic mRNA contains a cw-acting terminator before the first structural gene. When tryptophan levels are high the ribosomes read straight through this short ORF and the RNAP terminates transcription at the terminator where the RNA has formed a hairpin structure. When tryptophan levels are low the ribosomes pause within the short ORF of the leader mRNA due to lack of charged tR N A ^. This allows the mRNA to fold into an alternative secondary structure and prevents the formation of the hairpin structure, allowing transcription to continue into the tip operon (Landick et al., 1996).

Non-processive antitermination or simply antitermination of the E. coli bgl and B. subtilis sac catabolic opérons involves a similar mechanism whereby a regulatory protein interacts with the leader mRNA sequences to prevent the formation of a terminator loop. Since both of these systems are inducible each contains an additional component to regulate the activity of the antitermination factor and this will be discussed at a later stage (section 1.4.1).

Chapter 1: INTRODUCTION

1.3.5.2 Antitermination in the E. coli bgl operon

The bgl operon of E. coli specifies the enzymes involved in the catabolism of aromatic P-glucosides such as arbutin and salicin. The operon, which is cryptic and uninducible in wild-type strains (Mahadevan et a/., 1987), is activated by spontaneous mutations that cause an increase in the activity of the normally weak promoter (Schnetz and Rak, 1988;

1992). The bgl operon consists of three genes preceded by a cAMP/CAP dependent promoter (Figure 1.4). The first gene of the operon, bglG, encodes the transcription antiterminator protein and is flanked by two rho-independent transcriptional terminators which share a highly conserved sequence motif, of approximately 30 nucleotides, proximal to and extending into their stem-loop structures (Schnetz et al., 1987). This sequence motif or ribonucleic antiterminator (RAT) has been shown to be bound specifically by BglG to cause antitermination (Houman et al., 1990). The second gene of the operon, bglF, encodes a p-glucoside-specific transport protein, enzyme H®®' (En®®*). This forms part of the phosphoenolpyruvate (PEP) sugar-phosphotransferase system (PTS) and phosphorylates ligand-substrates in conjunction with their transport (Schnetz et al., 1996). BglF also functions as a negative regulator of operon expression by regulating the activity of BglG (Mahadevan et al., 1987). The mechanism of regulation exhibited by BglF will be covered in section 1.4.1.1. bglB encodes the catabolic activity, a phospho-P-glucosidase (Prasad and Schaefler, 1974).

1.3.5.3 Antitermination in the B. subtilis sac opérons

In B. subtilis, two systems involved in sucrose metabolism are controlled by

antitermination and the corresponding anti terminator proteins as well as the terminator structures have a high degree of similarity to those of the E. coli bgl operon. Sucrose induces the synthesis of at least three proteins in B. subtilis, an intracellular phosphosucrase {sacA), an extracellular levansucrase {sacB) and a sucrose specific transport protein, enzyme 11^^ (Ell^^), {sacP) which is part of the PTS (Steinmetz et al.,

1989). The DNA sequence upstream of the sacPA operon contains a transcription termination sequence almost identical to that found upstream of the sacB gene (Arnaud

et al., 1992) and both leader transcripts contain RAT sequences to which the

antiterminator proteins, SacT (sacPA operon) and Sac Y (sacB operon) bind in the presence of sucrose (Aymerich and Steimnetz 1992; Arnaud et al., 1996) (Figure 1.5).

T O

bglG

o

bglF

bglB

Figure 1.4 Structural organization o f the bgl operon o f E. coli.

The CAP/cAMP dependent promoter (p) and rho-independent transcriptional terminators (T) are shown.

sacT

sacP

sacA

B

o

sacB

sacY

sacX

Figure 1.5 Structural organization o f the sac opérons o f B. subtilis.

(A) The sac PA operon. (B) The sacB operon. The promoters (p) and rho-independent transcriptional terminators (T) are shown.

Chapter 1: INTRODUCTION

sacY itself is part of the sacS locus which also contains the sacX gene, which encodes a protein homologous to SacP (56% identity) and to several other EII^^ (Rutberg, 1997). BglG, SacY and SacT all belong to the same family of bacterial transcriptional antiterminators (van Tilbeurgh et oA, 1997). The RAT sequences of sacPA and sacB

differ from each other at only three positions and in vitro mutagenesis has been used to show that these differences are responsible for the specific induction of sacPA by SacT and sacB by SacY (Aymerich and Steinmetz, 1992). Aymerich and Steinmetz (1992) have shown that mutations increasing the similarity of the sacB RAT sequence to those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively, but that most of these changes did not strongly affect induction by SacY. Cross talk between the two regulatory systems controlling the sacPA and sacB genes has been observed under certain conditions (Arnaud et al., 1992).

1.3.5.4 SacY and BglG: Sequence specific RNA binding proteins

SacY and BglG function by binding the RAT sequences of the sacB or bgl mRNA’s which partly overlap the terminator sequences located upstream of the sacB and bgl

coding regions (Houman et al., 1990; Aymerich and Steinmetz, 1992). The leader region DNA sequences contain an axis of dyad symmetiy such that the transcript will preferentially adopt a stem-loop secondary structure. The formation of this secondary structure in the mRNA causes the RNA polymerase to terminate transcription. Under inducing conditions, SacY and BglG must bind to the nascent mRNA before the formation of the terminator structure. Recent experiments with truncated forms of both proteins show that the N-terminal fragments of SacY, (1-55), and BglG, (3-58) have RNA binding activities of the efficiency and specificity similar to that of the full-length proteins (Manival et al., 1997). This shows that SacY and BglG have their RNA binding domains within the N-terminal region. Additionally it has been shown that the

sacB RAT sequences can adopt an alternative folded back stem-loop structure which is rather unstable, and the presence of SacY(l-55) stabilizes this RAT structure. The formation of the RAT stem-loop then excludes the formation of the terminator structure.

SacY(l-55) is folded as a symmetrical dimer in solution, as well as in various crystal forms (van Tilbeurgh et al., 1997) and is not disrupted upon RAT binding, suggesting that in vivo, the full-length active form of the SacY protein is also a dimer (Manival et

al.y 1997). Within the SacY dimer four-stranded antiparallel p sheets of each monomer face each other in a roughly perpendicular orientation to form an eight-stranded p barrel covered on both sides by the loops joining strand 3 and strand 4 of each monomer (Figure 1.6). NMR titration of complex formation between SacY(l-55) and the RAT target indicates that the amino acids involved in RNA recognition are located close to the dimer interface. The dimer can be viewed as a molecular clamp, stabilizing the RAT structure in order to prevent the formation of the terminator structure (Aymerich and Steinmetz, 1992; Manival et at., 1997; van Tilbeurgh et al.y 1997).

It has been shown that under inducing conditions proteins such as BglG, SacT or SacY cause antitermination by binding close to transcription terminator sequences to prevent the formation of the terminators and allow the RNA polymerase to transcribe the downstream genes. Without a secondary control element transcription antitermination systems would lead to constitutive operon expression. In the bgl operon BglF acts as a negative regulator by controlling the activity of BglG and the activity of SacY is regulated by SacX in a similar manner. These regulatory systems will be described in the following section.

1.4 Two component signal transduction in