Biology Reference
In-Depth Information
Gene expression studies on the eight genes of Rbps of
A
.
variabilis
M3 revealed that after a
temperature shift from 38°C to 22°C, the transcripts of
rbpF
attained maximum levels at the fi rst hour
followed by the
rbpE
transcripts at the second hour. However, the mRNA levels of
rbpC
,
rbpA3
,
rbpE
and
rbpF
have been found to be transient. The gene transcripts of
rbpA1
and
rbpA2
were found to be
high at steady state levels at the low temperature. However, mRNA levels of the former gene reached
their maximum level within 3 h whereas the transcripts for the latter gene attained maximum levels
within 2 h. The levels of these transcripts for these two genes declined gradually but when transferred
back to 38°C these were barely detectable. There was a corresponding increase in the levels of Rbp
proteins during cold shock. Two of the genes
rbpB
and
rbpD
were expressed more or less constitutively
(Sato, 1994, 1995; Maruyama
et al
., 1999). Sato and Murayama (1997) identifi ed that the transcription of
rbpA3
is driven by two promoters. One of the promoters is active at high temperatures and its activity
is suppressed at the low temperatures. In contrast, the activity of the other promoter increased after
a temperature shift down from 38°C to 22°C. Further, to know the involvement of 5'-untranslated
region (5'-UTR) in the cold regulation of the
rbpA1
gene, a series of deletions of the
rbpA1
gene were
transcriptionally fused to
lacZ
promoter and then the constructs were introduced into
A
.
variabilis
M3.
Additionally, a series of plasmids based on shuttle vector pRA101 were fused with short DNA fragments
in the 5'-UTR region of the
rbpA1
gene and
lacZ
as reporter gene. These experiments revealed that short
DNA fragments one from -139 to +201 and another from -139 to +149 both conferred cold regulation
on the
lacZ
gene. Both transcripts of
lacZ
gene and the β-galactosidase activity increased at the low
temperature. Sato and Nakamura (1998) concluded that DNA fragment from -139 to +140 conferred
high constitutive expression of gene
rbpA1
at 38°C as well as 22°C. This indicates the constitutive
nature of the promoter of the
rbpA1
gene and the 5'-UTR sequence is required to reduce transcription
of the
rbpA1
gene at high temperature. At high temperatures the expression of this gene is repressed
by the binding of repressor protein(s) to 5'-UTR region of the
rbpA1
gene. Gel mobility shift assays and
affi nity purifi cation of DNA-binding proteins revealed the formation of two complexes. There are two
polypeptides (75 and 32 kDa) that are involved in the formation of complex 1 and one polypeptide
(72 kDa) is involved in the formation of complex 2 (Sato and Nakamura, 1998). Maruyama
et al
. (1999)
compared the 5'-UTR of the
rbp
genes and found four conserved regions. The fi rst one is the conserved
ribosome-binding site (RBS; 5'-TTCGGAGA-3') that is located about 6 bases upstream of the translation
initiation codon. The conserved box I (5'-TCTCCGAA-3'), box II (5'-TTTGTTTNNAGT-3') and box III
((5'-TTCGGYGA-3') are located about 40, 90 and 110 bases upstream of the initiation codon, respectively.
It is interesting to note that the RBS is complementary to box I and box III is complementary to box I.
Phylogenetic analysis of Rbp proteins revealed that Rbps of cyanobacteria and glycine-rich proteins
(GRPs) of eukaryotes do not belong to the same lineage but the Rbp proteins of cyanobacteria have
been suggested to have diverged from the GRPs. The apparent similarities in both structure and
regulation between the cyanobacterial Rbp proteins and eukaryotic GRPs may have been due to
convergent evolution.
RNA-binding properties of Rbp proteins of
Anabaena
sp. strain PCC 7120 have been characterized
by Hamano
et al
. (2004). Based on the comprehensive genomic data, the existence of two classes
of Rbp proteins (Classes I and II) has been proposed that diverged before the diversifi cation of
cyanobacteria. Class I Rbp proteins are further classifi ed into two types, i.e. with or without a
C-terminal glycine-rich domain. RbpA1 belongs to the Class I Rbps with high affi nity to C-rich and
G-rich sequences. The Class II Rbps have low affi nity to G-rich sequences. Site-specifi c mutagenesis
analysis revealed that aromatic amino acids Tyr4 and Phe46 are very important in RNA-binding as
well as secondary structure maintenance. However, it is also suggested that the C-terminal glycine-
rich domain itself does not contribute to the RNA-binding but Arg83 located close to C-terminal end