INTRODUCTION

The physiological function of haemoglobins
in species other than vertebrate animals has not been well established.
Although
the role of vertebrate haemoglobins as facilitators of oxygen diffusion is well
established, the function of globins in invertebrate animals, as well as in
plants, protozoa, fungi, and bacteria, is generally unclear. The present
data suggest that, despite the high degree of homology between globins of
prokaryotic and eukaryotic microorganisms, each species may have developed a
discrete role for its hemoglobin.1

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There has been extensive comparative research of the
haemoglobins of micro-organisms-yeast, bacteria, invertebrates/vertebrates
(mammals), and humans; to identify the presence similar parallel oxygen-sensing
systems in all organisms and understand the functions of these highly conserved
haemoglobin proteins. The aerobic bacteria, Vitreoscilla single-domain haemoglobin
(VtHb) has been extensively studied in this regard.

Whereas most vertebrate haemoglobins
are composed of two types of polypeptide subunits, ? and ?, which have single
heme domains and form ?2?2 tetramers; invertebrate and microbial haemoglobins
are more varied. Many of the bacterial and fungal haemoglobins that have been
characterized to date fall into two general categories: dimeric hemoproteins
composed of two single heme domain polypeptides and monomeric flavohaemoproteins
composed of a single polypeptide containing a single heme-binding domain and a
single Flavin-binding domain.2 These flavohaemoglobins
(flavoHbs) are made of a globin domain fused with a ferredoxin reductase-like
FAD- and NAD-binding modules. These proteins are widely represented among
bacteria and yeasts3

 

 

GENE
REGULATION

Haemoglobins recently sequenced
from the bacteria E. coli and Alcaligenes eutrophus and the yeasts Saccharomyces cerevisiae (baker’s
yeast) and Candida norvegensis are two-domain proteins with nearly 40% sequence
identity. The flavohaemoproteins contain both
heme and flavin binding domains and which are capable of transferring electrons
from NADPH to heme iron.2  Their N-terminal regions share
substantial sequence homology with the single-domain of the aerobic bacteria
Vitreoscilla (VtHb), whereas the C terminus contains a reductase domain with
potential binding sites for flavin (FAD) and NADPH. 4  

Baker’s yeast is a facultative
aerobe, capable of growth in the complete absence of molecular oxygen, conditions
under which yeast cells can grow but under which most mammalian cells, let
alone the intact organism, could not long survive.5

The S. cerevisiae flavohaemoglobin
gene (YHB) has been mapped to chromosome 7, near the formyltetrahydrofolate
synthase (ADE3) locus. The genes involved in S. cerevisae oxygen regulation are
divided into two broad categories- (1) genes regulated by heme and (2) genes
whose regulation is heme independent.

Heme-regulated genes fall into two
classes: heme-activated and heme-repressed genes. Activation of the
flavohaemoglobin gene is achieved through one of two transcriptional
activators, the heme-dependent HAP1 protein or the heme-activated,
glucose-repressed HAP2/3/4 complex.6

S. cerevisiae globin message is
induced during logarithmic growth and under oxygen-replete conditions. YHB1 is
expressed in hyperoxic states and conditions that promote oxidative stress.
These findings suggest that YHb plays a role in the oxidative stress
response in yeast functioning as an oxygen scavenger. 2

YHb is not essential for cell
growth. Normally, actively respiring yeast cells have very low levels of the flavohaemoglobin
and flavohaemoglobin gene disruption does not alter cell viability or growth in
a variety of oxygen conditions and carbon sources. This protein binds oxygen
reversibly only when NADPH is present, indicating that it has an NAD(P)H
reductase activity for the heme domain. However, when the mitochondrial
electron transport chain has been compromised by either mutation (i.e. the
deletion of the mitochondrial genome) or respiration inhibitors (e.g. antimycin
A)- the expression of the flavohemoglobin gene, YHB1 is upregulated.2

Saccharomyces cerevisiae also
expresses several isozymes of cytochrome c oxidase. Isozymes
which incorporate the Vb isoform have both higher turnover rates and higher
rates of heme oxidation than isozymes which incorporate Va.
YHB1 level has been shown to increase in cells engineered to express the
hypoxic isoform-Vb, of cytochrome c oxidase subunit V under aerobic conditions.

Regulation of the S. cerevisiae
flavohemoglobin gene is clearly controlled by cell density and oxygen tension
but in a manner different from that of the bacterial globins previously
studied. There is Repression of YHG mRNA with increasing cell density.

The repression of endogenous YHG
gene expression upon exit from exponential phase may be due to a combination of
factors such as hypoxic conditions, induction of a repressor, or the altered
chromatin structure at higher culture densities that has been implicated in
repressing most exponential phase mRNA species

 

In S. cerevisiae there are two
classes of yeast genes: “aerobic” genes (e.g. HMG1, COX5a, CYC1, AAC1, AAC2,
TIF51a) , which are expressed optimally in the presence of air, and “hypoxic”
genes (e.g. HEM13, ERG11, HMG2, CPR1, SUT1, OLE1, COX5b, CYC7, AAE3, ANB1,
TIF51b), which are expressed optimally at low oxygen concentrations2

S. cerevisae flavohaemoglobin (YHG)
transcriptional activation is modulated by oxygen concentration. Below 0.1%
oxygen concentration aerobic activation of the YHG promoter ends and the
anaerobic expression of the YHG message begins. Transcript levels for YHB1 are
reduced in the absence of oxygen (lane 1) and slightly elevated under hyperoxic
conditions (lane 3). Thus, 0.1% likely straddles the concentration of oxygen
necessary for heme biosynthesis.

                                                            Increasing
oxygen tension

Oxygen availability is sensed through
cellular heme levels. Consequently, under hypoxic conditions, heme levels are
reduced. 5  In S. cerevisiae, transcription factors
involved in oxygen regulation of gene expression have been well characterized. Aerobic
expression of YHG is predominantly activated by the HAP1 and HAP2/3/4
transcription factor complexes. An anaerobic system to produce YHG message
independent of the HAPs is also present. Although there is a decrease in YHG
gene expression as the cells approach anaerobiosis.

Hap1 is a
protein composed of a zinc finger DNA binding domain at the N-terminus, a heme
binding domain within the central region, and a transcriptional activation
domain at the C-terminus.5 YHG
promoter/lacZ fusion construct is also regulated by heme and the HAPs

TIF51A/B gene pair also regulate
heme biosynthesis. Under aerobic conditions, heme accumulates and serves as an
effector for the transcriptional activator Hap1. The heme-Hap1 complex
activates transcription of the ROXI gene that encodes a repressor of one set of
hypoxic genes. For example, above 0.1% Oxygen concentration, only TIF51A is
present; below this concentration, only TIF51B can be detected

YHG is probably not involved in
facilitating oxygen storage or diffusion during hypoxic electron transport.
Contrary to many other genes involved in respiration, the YHG message
represents the first known example of a HAP2/3/4-regulated gene that is not
glucose-repressed, indicating that the gene may be required in both fermentable
and non-fermentable carbon sources. There is an increase in YHG mRNA levels in
cells grown under high oxygen tension. This phenomenon can be attributed to
increased heme levels, which also stimulate superoxide dismutase and catalase
transcription in S. cerevisiae. Therefore, flavohemoglobin in S. cerevisiae may
also be involved in the detoxification of oxygen.

Cells sense hypoxia through the
inability to maintain oxygen-dependent heme biosynthesis. Under hypoxic
conditions, heme levels fall, and a heme-deficient Hap1 complex represses ROX1
expression. When heme
is bound, Hap1 acts as a transcriptional activator of genes containing its
recognition site (5’CGGN6CGG) -for the most part, genes encoding a variety of
respiratory and oxidative stress functions i.e. aerobic genes.

In addition, the ROX1 gene encoding the repressor of hypoxic
genes is also activated by the Hap1-heme complex. The Rox1 repressor binds to
its cognate site upstream of the hypoxic genes to repress their transcription.
Under hypoxic or anaerobic growth conditions, heme levels are reduced. Hap1
still binds to its cognate site, but in the absence of heme, additional
proteins bind to Hap1 creating a larger complex that represses transcription. HAP1
directly represses its own transcription by binding HAP1 promoters. Consequently,
under hypoxic conditions, ROX1 expression is repressed resulting in the
derepression of the hypoxic genes. Therefore, heme is the effector molecule.

 

Rox1 consists of 368 amino acids that can be divided into
three domains. The first third of the protein consists of an HMG DNA binding
motif. This motif is found in a number of DNA binding and bending proteins
including both sequence-specific regulatory proteins and general DNA binding
chromatin proteins. The ability of these proteins to bend DNA has led to their
designation as architectural proteins, proteins that induce conformations in
DNA that may be important for their function. Using gel retardation and DNase protection studies, this
domain was found to bind as a monomer to the previously mapped operator sites
in the ANB1 gene. Experiments with chimeric proteins suggests that this bend is
not essential but enhances repression activity.

The Rox1 protein consists of at least two additional
domains. There is a glutamine-rich region following the HMG domain, extending
from residues 100 to 123. Deletion of this region does not affect either the function
or the lability of Rox1, and its purpose is not known. The remainder of the
protein represents the repression domain. Deletion of this domain results in a
DNA binding protein with no repression activity, and fusion of this domain to a
heterologous DNA binding protein (the amino terminus of the yeast protein Ga14)
results in a chimeric protein with repressor activity.

The binding of transcriptional activators often lies
hundreds of base pairs from the TATA box, where the basal transcriptional
machinery and RNA polymerase II bind. Many genes have multiple, differentially
regulated activators that can act individually and additively to set
transcription levels. Such promoters preclude repression mechanisms that simply
act through competitive binding of the repressor with an activator or RNA
polymerase, and suggest that repression must be an active process. Rox1 binding to DNA alone is not
sufficient to repress hypoxic gene transcription. A general repression complex consisting
of two proteins, Tup1 and Ssn6, is also required which functions via the HOG-MAP
kinase signalling pathway.

 

CONCLUSION

As in all organisms, gene
expression in S. cerevisae is a complex process. Despite the highly conserved nature
of the globin gene among many species including man, and the extensive
sequence homology between baker’s yeast and Candida as well as those of several
bacterial species, the
S. cerevisiae flavohemoglobin gene (YHB1) may have distinct transcriptional
activation mechanisms with consequent unique function for its flavohemoprotein
(YHb). 1

S. cerevisiae YHb is an important
regulator of many other genes, regulating the expression and transcription of
several genes based on oxygen concentration in hypoxic or aerobic environments.
The  S. cerevisiae heme activator protein
Hap1 binds to DNA and activates transcription of genes encoding functions required
for respiration and for controlling oxidative damage, in response to heme.7  

 

 

 

BIBLIOGRAPHY

1.        Crawford MJ,
Sherman DR, Goldberg DE. Regulation of Saccharomyces cerevisiae flavohemoglobin
gene expression. J Biol Chem Internet. 1995 Mar 24;270(12):6991–6.

2.        Zhao XJ, Raitt
D, V Burke P, Clewell AS, Kwast KE, Poyton RO. Function and expression of flavohemoglobin
in Saccharomyces cerevisiae. Evidence for a role in the oxidative stress
response. J Biol Chem Internet. 1996 Oct 11;271(41):25131–8.

3.        Bonamore A,
Boffi A. Flavohemoglobin: Structure and reactivity. IUBMB Life Internet. 2007
Dec 12;60(1):19–28.

4.        Zhu H, Riggs
AF. Yeast flavohemoglobin is an ancient protein related to globins and a
reductase family. Biochemistry Internet. 1992;89:5015–9.

5.        Zitomer RS,
Carrico P, Deckert J. Regulation of hypoxic gene expression in yeast. Kidney
Int Internet. 1997;51(2):507–13.

6.        Zitomer RS,
Lowry C V. Regulation of gene expression by oxygen in Saccharomyces cerevisiae.
Microbiol Rev Internet. 1992 Mar 1;56(1):1–11.

7.        Hon T, Lee HC,
Hu Z, Iyer VR, Zhang L. The Heme Activator Protein Hap1 Represses Transcription
by a Heme-Independent Mechanism in Saccharomyces cerevisiae.

 

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