A Brief Overview of LacI-Family Transcriptional Regulators in Bacteria

Abstract

Various regulatory mechanisms control the initiation of transcription to regulate gene expression. Several families of transcription factors (TFs) regulate this biological process. The lactose repressor protein (LacI) family is the second-largest family of TFs in bacteria and archaea. Members of the LacI family have been reported to regulate gene transcription in Enterobacteria, Proteobacteria, and Firmicutes. This family has more than 1,000 members that have been characterized, as well as many putative homologs. In terms of their characteristic function, the LacI family members are global regulators of sugar metabolism. This family perceives sugar effectors and, in response, controls the expression of the genes involved in the use of carbohydrates. Some members of the LacI family control the expression of specific genes and act as repressors or activators, although the majority of LacI family members are repressors. The presence of an inducer and the location of the operator, whether upstream (O3) or downstream (O1, O2) of the target gene, affect the LacI regulation mechanism. The interaction between the operator and LacI relies on the helix-turn-helix (HTH) motif in the LacI protein. The (T)G-A-CG-T-C(A) sequence in the operator, which contains the conserved core CG group, is recognized by LacI family TFs as the critical motif.

1. Introduction

Organisms possess regulatory systems for gene expression, ensuring that the genes expressed align with the cell's requirements during certain growth conditions. Therefore, the expression of different types of genes is important for the survival of organisms. The control of gene expression influences how genetic information is transformed into different biological products. Gene expression is regulated according to the needs of the cells [1]. Certain gene products are required under particular growth conditions but are not necessarily required under all circumstances. Genes are not expressed when their products are not needed, but may be expressed at low to high levels in response to certain conditions or cues. The gene expression will be upregulated when the cell meets an environment that requires a certain amount of gene product [1, 2].

In 1961, Jacob and Monod described the key concepts of transcriptional control in bacterial systems. They stated that the regulation of genes encompasses three distinct stages in the process of generating the product of the functional gene. Primarily, gene transcription can be subject to regulation, a phenomenon termed transcriptional regulation. The number of gene transcripts directly impacts the abundance of the product of the gene. Secondly, in the case where the gene codes for a protein, regulatory mechanisms can operate at the translational level, referred to as translational regulation. Here, the frequency of mRNA translation governs the output of the gene product. Lastly, the production of gene products can be modulated after the transcription step (post-transcriptional) or translation step (post-translational) [1].

Gene regulation at the transcription level is controlled by transcription factors (TFs), which are DNA-binding proteins that repress or activate transcription by binding to a specific DNA sequence within the genome. Transcription factors build genetic circuits that regulate gene expression. In bacteria, the initiation of transcription is regulated via two distinct mechanisms: (1) The promoter‑centric mechanism, in which specific TFs interact with the promoter to alter its ability to bind RNA polymerase (RNAP); and (2) the RNA‑centric mechanism, in which TFs interact with RNAP to alter its promoter preference [3].

The regulation of gene expression is the most prevalent at the onset of transcription. The regulatory mechanism involves accessory proteins that affect the binding of RNAP at the promoter site. There are two mechanisms by which accessory proteins regulate the promoter site: positive regulation by activators and negative regulation by repressors [1]. Operons are a type of gene structure in which the operator is located right next to the promoter, although the regulator controls the actual functioning of the operon [4].

Repressor proteins are the main type of regulator of gene expression in bacteria because the expression of bacterial genes is generally “on” by default [6]. RNA polymerase is the enzyme that recognizes all promoter sequences in bacterial genomes and transcribes all the genes in the organism. However, if repressor proteins are present, they prevent RNAP from binding to the promoter sequence [5]. Transcription is most commonly induced by removing repressor proteins to enable the binding of RNAP and the transcription of downstream genes [7].

Lewis [8] described a group of allosteric DNA-binding regulators known as the lactose repressor protein (LacI) family transcriptional regulators, which have conserved amino acid sequences. The majority of the known TFs in the LacI family sense sugar effectors and control the expression of genes related to the use of carbohydrates [9]. Weickert and Adhya first demonstrated LacI in Escherichia coli as the model system for studies on bacterial transcriptional regulation in 1992 [10]. According to a 2008 BLAST search of Swiss-Prot [11], the LacI family has more than 1,000 characterized sequences and many hypothetical homologs. The LacI family plays a vital role in numerous bacterial species. In the phylum Firmicutes, these TFs are responsible for the preferential usage of specific sugars over others. Members of the LacI family are known to be involved in both the repression and activation of catabolic genes in the presence or absence or a related substrate and/or a preferred substrate [12,13,14].

Although there have been many studies on the LacI family, there is still much to learn about their main functions and mechanisms. In this review, we discuss some of the main findings of studies on the LacI family. We summarize the current state of knowledge about this family of transcriptional regulators in bacteria and their function, mechanism, and structure.

2. Members of the LacI family

At first, it was reported that the LacI family had more than 21 members [10, 15]. However, as research progressed, more members of the LacI family were identified. According to the InterPro database (https://www.ebi.ac.uk/interpro/entry/cdd/CD01392/), the LacI family has more than 500 members. A 2008 BLAST search of Swiss-Prot identified more than 1,000 members distributed among almost all types of bacteria. Ravchhev [9] identified 1,300 LacI TFs from 270 bacterial genomes based on searches of homologous DNA-binding patterns and putative target genes.

The mechanism by which proteins recognize DNA sequences is an important part of the gene regulation process. A protein can be categorized as a member of a particular family if it has certain similarities or resemblances when interacting with DNA. Members of the LacI family can perceive sugar effectors and trigger the expression of genes encoding proteins involved in sugar metabolism [9]. They can function as either activators or repressors of gene transcription.

Orthologous protein sequences have the same molecular characteristics. Similarly, synteny-conserved gene order was revealed to be an excellent indicator of functional equivalence [16,17,18,19]. Most members of a family interact with a limited number of operators in the genome. For example, LacI in E. coli solely represses the lac operon in the absence of lactose. This raises the question as to how bacteria use homologous TFs to regulate the expression of one or a few genes/operons (local regulation) or many genes/operons (global regulation) [19] (Table 1). According to Ravcheev’s findings [9], almost 90% of LacI family members are local regulators, but 125 LacI family members are global regulators, such as CcpA in Firmicutes, FruR in Enterobacteria, the purine repressor protein (PurR) in Gammaproteobacteria, and GluR, GapR, and PckR in Alphaproteobacteria.

3. Genes regulated by LacI family members

The genes that are regulated by TFs are known as regulons [20]. Regulons are groups of genes that are all required for a particular process but are physically situated in various sections of the chromosome and have their own promoter(s). The promoters of all the genes in a regulon are controlled in the same way, allowing for coordinated production of the required gene products.

Regulons can be identified by several methods. For example, in Lactobacillus plantarum, regulons were identified on the basis of the structural similarity of the DNA motifs recognized by LacI TFs [19], as were regulons in Dickeya dadantii [21]. Swint-Kruse and Matthews [11] identified that the structural characteristics of the fold in LacI TFs affects their interactions with DNA and ligands, and associations between dimers.

The relationship between a protein and DNA is largely determined by the specificity of the recognition site. Francke [19] classified orthologous TFs into groups that share the same gene context to produce putative groups of orthologous functional equivalents and correctly predicted motifs of LacI TFs with high specificity. Hars and Matthews [22, 23] stated that van der Waals contacts and multiple hydrogen bonds between the protein’s side chains and the edges of the base pairs exposed in the major groove of the DNA specified the binding sites in the DNA sequence. This is the basis of target DNA recognition by TFs for control. LacI-family regulons have been studied in detail, and critical information was summarized in Table 1.

Table 1: Members of LacI family of transcriptional regulators and their regulated genes

LacI Family Member	Examples of Regulated genes	Function	Origin	Reference
PurR	glyA, gcv, prs	Repression of glycine synthesis	Escherichia coli	[24, 25, 26, 27]
	purF, purD, purEK, purB, purC, purL, purMN	Repression synthesis of inosine monophosphate (IMP)
	guaBA	Repression of conversion of IMP to guanosine monophosphate (GMP)
	pyrC and pyrD	Repression of synthesis of pyrimidine nucleotides
FruR	pckA, edd, gapB, mtlA, pykF	Repression of modulation the direction of carbon flow	Escherichia coli	[28]
CcpA	amyE, amyO	Repression of α-amylase synthesis	Bacillus subtilis	[29, 30, 31, 32, 33]
	alsS, alsD, alsR	Activator of acetoin biosynthesis	Lactobacillus casei ATCC 393 (American Type Culture Collection, USA)
	ackA	Activator of acetate secretion
	lacTEGF	Regulation of catabolite repression of N-acetylglucosaminidase and phosphor-β -galactosidase
CcpB	gntR, gntK, gntP, gntZ, xyl	Regulation of catabolite repression	Bacillus subtilis	[34]
DegA	iolX	Repression of NAD+ -dependent scyllo-inositol dehydrogenase	Bacillus subtilis	[35]
RbsR	rbsK	Regulation of controlling ribose transport	Escherichia coli Bacillus subtilis Proteobacteria Corynebacterium glutamicum ATCC 13032 Bifidobacterium Serratia marcescens Db11 Dickeya dadantii, Pectobacterium atrosepticum, Erwinia amylovora, Yersinia enterocolitica Klebsiella pneumoniae	[36, 37, 38]
	rbsA	Regulation of ATPase
	rbsB	Regulation of periplasmic binding protein
	rbsC	Regulation of membrane permease
	rbsD	Regulation of ribose mutarotase
	rbsR	Regulation/ repress rbsDACBKR operon
CytR	udp comEA pilA chiA-1	Repress uridine phosphorylase	Vibrio cholerae	[39, 40]
	cdd	Repress nucleoside uptake and catabolism in nucleoside-poor environments	Vibrio cholerae Escherichia coli
RegA	c2, cy, bc1, bc1	Activator for cytochromes (photosynthesis process)	Rhodobacter capsulatus	[41, 42]
GalR	galR-galK intergenic region	Repress galactose metabolism	Streptococcus mutans Escherichia coli	[43, 10]
GalS	galP	Repress galactose permease metabolism	Escherichia coli	[44]
	mgl	Repress β-methylgalactoside transport system
MalR	malXCD	Regulation of maltosaccharide uptake	Streptococcus pneumoniae	[45]
	malMP	Regulation of maltosaccharide utilization
YjgS(Syn: GntH, IdnR)	gntT	Regulation of gluconate transport	Escherichia coli Escherichia coli K-12	[46, 47]
OrfA	IS3	Inhibitor of IS3 transposase	Escherichia coli	[48]
	CD134	Repression of viral replication and infection in T cells	Feline immunodeficiency virus (FIV)	[49]
TreR	treB treC	Repressor of operons involved in trehalose transport and degradation under osmotic stress	Escherichia coli	[50, 51]
LacI	lacA	Repression of galactoside O-acetyltransferase	Escherichia coli str. K-12 substr. MG1655	[52, 53, 54, 55]
	lacY	Repression of lactose permease, MFS family	Escherichia coli str. K-12 substr. MG1655 Klebsiella pneumoniae subsp. pneumoniae MGH 78578
	lacZ	Repression of β -galactosidase
LacR	lacR	Lactose utilization transcriptional repressor	Lactobacillus plantarum WCFS1	[56]
	lacZ	Repression β-galactosidase
	lacS	Repression lactose and galactose permease, GPH translocator family
MalI	malI	Maltose regulon regulatory protein MalI	Klebsiella pneumoniae subsp. pneumoniae MGH 78578 Escherichia coli K-12	[57] [58]
	malX	PTS system, maltose and glucose-specific IIC component / PTS system, maltose and glucose-specific IIB component
	malY	Cystathionine β -lyase/ Maltose regulon modulator
XylR	xylAB/xylR	Regulation of xylan and xylose utilization	Bacillus subtilis Bacillus halodurans Clostridium difficile	[59, 60, 61]
	xynCB	Regulation of β-xyloside permease and β-xylosidase	Bacillus subtilis Clostridium acetobutylicum
	ygaE	Regulation out membrane proteins ompf/ompc at the early stage of hyperosmotic stress	Bacillus subtilis
	xylAB	Regulation of xylose isomerase (xylA) and xylulose kinase (xylB)	Bacillus stearothermophilus Thermoanaerobacter ethanolicus Lactobacillus brevis
	xylR	regulation of xylose utilization	Escherichia coli.coli Bacillus. stearothermophilus Clostridium acetobutylicum
	xyn1	regulation of xylose	B. stearothermophilus Bacillus sp.
	xylS-pts1-2-3-4	Regulation of xylosidase and a PTS transport system	Clostridium difficile Clostridium difficile
	xylR/xylS-pts1-2-3-4-xylAB
CscR	cscB	Regulation of sucrose permease, major facilitator superfamily	Bifidobacterium gallicum DSM 20093 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)	[62]
	cscA	Regulation of sucrase (EC 3.2.1.26)
ScrR	scrA	Repression of sucrose specific PTS system, IIABC component	Clostridium beijerincki NCIMB 8052 (National Collections of Industrial Food and Marine Bacteria, UK)	[63, 64]
	scrR	Repression of sucrose utilization transcriptional regulator ScrR	Staphylococcus aureus subsp. aureus N315
	scrB	Repression of sucrose-6-phosphate	Clostridium beijerincki NCIMB 8052 Clostridium beijerincki NCIMB 8052
	scrK	Repression of fructokinase
EbgR	ebgR	Evolved β -D-galactosidase transcriptional repressor	Vibrio angustum S14	[65]
	ebgA	Evolved β -D-galactosidase, alpha subunit
	ebgC	Evolved β -D-galactosidase, β subunit
	ygjl	Evolved β -galactoside transporter, AAP family
AraR	araABDLMNPQ-abfA, araE and araR ydjK xsa abnA	Repressor of the utilization of arabinose	Bacillus subtilis	[59, 66]
	abfA-araM araDBA-xsa		Bacillus halodurans
	ptk (CAC1343) abf2-CAC1530		Clostridium acetobutylicum
	araK-araDA/araE araR bfA		Enterococcus faecium
BfrR	bfrB	Regulation of putative fructooligosaccharide ABC transporter, substrate-binding component	Bifidobacterium adolescentis ATCC 15703	[67]
	bfrC	Regulation of putative fructooligosaccharide ABC transporter, permease component 1
	bfrD	Regulation of putative fructooligosaccharide ABC transporter, permease component 2
	bfrR	Regulation of putative transcriptional regulator of fructooligosaccharide utilization, LacI-family
	bfrA	Regulation of β-fructosidase
MsmR	msmR	Repress Transcriptional regulator of α-galactoside utilization	Bacillus amyloliquefaciens FZB42	[68]
	msmE	Repress Multiple sugar ABC transporter, stachyose-binding protein
	msmF	Repress Multiple sugar ABC transporter, membrane-spanning permease protein MsmF
	msmG	Repress Multiple sugar ABC transporter, membrane-spanning permease protein MsmG
	melA	Repress α-galactosidase
	nga	Activator for NAD-glycohydrolase	Streptococcus pyogenes	[69]
	nra/cpa	Activator for collagen-binding protein
	prtF2	Activator for fibronectin-binding protein F2 gene
NtdR	ntdR	Transcriptional regulator of neotrehalosadiamine utilization	Bacillus subtilis subsp. subtilis str. 168	[70]
	ntdA	Activator for Neotrehalosadiamine biosynthesis
	ntdB	Activator for Neotrehalosadiamine biosynthesis operon putative hydrolase
	ntdC	Activator for Neotrehalosadiamine biosynthesis operon putative oxidoreductase
YkvZ	bglAC	Regulation of aryl-phospho-β-D-glucosidases	Bacillus subtilis	[71]
LfaR	lfaR	Regulator for sugar utilization	Erwinia chrysanthemi 3937 Erwinia carotovora subsp. atroseptica SCRI1043	[21, 72]
	lfaT	Repress hexuronate-like transporter
	aguA	Repress α-glucuronidase, glycoside hydrolase family 31
LffR	lffR	Maltose operon transcriptional repressor MalR	Erwinia chrysanthemi 3937	[21]

4. Functions and mechanisms of LacI family members

Members of the LacI family function as global regulators in several bacterial lineages. To explore the thermodynamic and general mechanistic aspects of LacI regulators, equilibrium analyses were conducted using purified proteins, and their ability to bind to operator DNA and sugars was evaluated [10, 73, 74, 75]. There are three groups of sugar effectors; inducers, anti-inducers, and neutral effectors [76, 77]. The majority of sugars are inducers that decrease LacI’s affinity for DNA, thereby de-repressing gene expression. A few sugars are anti-inducers, which strengthen the binding of LacI to DNA, thereby further repressing gene expression. Allosteric regulation can be quantified as the difference in the affinity for one ligand in the absence and presence of saturating levels of the other ligand. Kinetics studies have provided insight into the time-scale of binding events and allosteric responses [78, 79, 80, 81]. Neutral sugar effectors bind specifically to a protein but have no functional effect. The function of LacI as a regulator of sugar metabolism was also reported by Ravcheev [9], who found that more than LacI ortholog groups controlled the catabolism of least 10 types of carbohydrates: sucrose, glucose and glucosides, galactose and galactosides, ribose, maltose and maltodextrins, inositol, gluconate and idonate, glucuronate and galacturonate, mannose and mannosides, fructose and fructooligosaccharides, and trehalose.

Lactose is converted into an isomer known as allolactose when it enters cells. It acts as an inducer to initiate the transcription of genes in the lac operon. Genes in the lac operon produce the proteins required for lactose breakdown. Lactose metabolism occurs when lactose is abundant but glucose is not. Lactose is naturally broken down by cells to produce energy. Isopropyl-D-thiogalactoside (IPTG), which functions similarly to allolactose, eliminates a repressor from the lac operon to promote gene expression.

In general, TFs have two fundamental roles; the ability to recognize and bind to short specific DNA sequences within regulatory regions, and the capacity to recruit or bind other proteins for transcriptional control [82, 83]. All LacI TFs have the same function — they bind to operator DNA and change how downstream genes are transcribed [84]. As mentioned before, regulatory proteins can function as positive or negative regulators depending on the operator’s position relative to that of the promoter, either upstream or inside/downstream of the target gene (Fig.1) [31, 85, 86, 87].

4.1 Positive regulators (activators)

Positive control occurs when genes are only expressed in the presence of an active regulator protein, such as an activator. When the positive regulatory protein is missing or inactive, the operon is turned off. de Crombrugghe [88] demonstrated that the ligand cyclic AMP (cAMP) triggered the binding of cAMP receptor protein (CAP) to target sites, and that the cAMP–CAP complex was necessary and sufficient for RNAP to initiate transcription at the lac operon promoter in E. coli. In addition to the lac operon, other operons whose genes encode products that metabolize particular sugars such as galactose, arabinose, and maltose are also influenced by CAP. Allolactose (or the unrestricted inducer IPTG and galactose) function as inducer effectors to relieve repression of gene expression.

When glucose levels are high, there is no need to express large quantities of the enzymes required for the digestion of lactose or other carbohydrates that give less energy than glucose [89]. When intracellular glucose levels are high, cAMP levels are low, and there are very few cAMP–CAP complexes. When intracellular glucose levels decrease and other carbohydrates must be digested, the cAMP levels rise. Increased cAMP levels indicate the presence of more cAMP–CAP complexes. The cAMP–CAP complex binds to a particular location on the DNA that is close to the promoter of lac operon genes as well as genes in other operons related to sugar metabolism [1]. Some LacIs such as CcpA, FruR, RegA, MsmR, and NtdA are positive regulators of gene expression.

4.2 Negative regulators (repressors)

Repressors coordinately regulate structural genes involved in lactose metabolism. The repressor protein has a high affinity for the lac operator in the absence of lactose, which makes it difficult for RNAP to bind to DNA, elongate transcribed RNA, or initiate gene transcription. The repressor prevents transcription from occurring until it comes into contact with an inducer chemical (such as lactose). Repression is mediated by the reversible interaction of the repressor with the operator, i.e., the regulatory component. The mRNAs of the structural genes, their encoded products, and the enzymatic activity of the products are produced when the repressor is stimulated [1].

LacI is necessary for the molecular mechanism of repressing the lac operon because it must bind to the operator DNA and respond to sugars as inducers. Once LacI binds to the operator site in the promoter, the RNAP cannot bind to the promoter, and gene transcription is blocked. The operator is a negative regulatory site that is bound by LacI. Brenowitz [90] revealed that a deletion mutant of LacI was unable to bind to DNA or form the tetramer protein.

Gene transcription is repressed by many members of the LacI TF family such as LacI, PurR, and the galactose repressor protein (GalR) [11]. When a repressor protein binds to a small metabolite, DNA binding and repression of gene expression are altered. PurR repression is enhanced by binding guanine or hypoxanthine. FruR and MsmR are thought to have repressor and activator activities, while nearly all LacI TFs are repressors.

Figure 1: Model describing the transcriptional regulation by TFs of LacI family. The binding and release of LacI from O1, O2 and O3 play a crucial role in controlling the transcription of the lac genes in response to the presence or absence of lactose and its derivatives as inducers. Different mechanisms function during various phases of the transcription cycle, and depending on the sigma factor involved [8, 91].

5. DNA-binding domain and motifs of LacI family members

5.1 Binding domains

The specificity of TFs for DNA binding is critical for gene regulation, and hence, for cellular function [92]. A DNA-binding domain (DBD) is an independently folded protein domain with at least one structural motif that recognizes a specific DNA sequence (a recognition sequence) or has a general affinity for DNA. Transcription factors bind to regulatory sequences in DNA and switch the transcription of genes on or off [93].

LacI is a tetramer comprising four protein subunits. According to Rutkauskas et al. [94], the tetramer is a tethered “dimer of dimers”, connected by a bundle of four alpha helices (Fig. 2) [8]. The monomer that makes up each subunit has three main parts: an N-terminal core (amino acids 1–60) with significantly lower affinity than that of the inducer-binding domain [95, 96]; a core domain (amino acids 61–330) that forms the dimer interface and binds to the inducer; and a domain at the C-terminal core (amino acids 331–360) that is responsible for creating the tetramer interface (6–9). This formation is a characteristic of the Venus fly-trap (VFT) family, which includes transport-related bacterial periplasmic binding proteins, the extracellular domain of G-protein coupled receptors, and other receptors that convert extracellular stimuli into intracellular signals. Proteins in the VFT family exhibit subdomain movements associated with function, and share structural homology in the core domain with LacI. LacI also has a short C-terminal domain required for the formation of the homo-tetramer and a hinge region between the DNA-binding and inducer-binding domains [97].

The protein–protein interaction between RNAP and TFs must be weak to allow for the frequent and rapid exchange of RNAP-interacting TFs. Effective protein–protein interactions rely on TFs binding to specific target sites close to the promoter, which raises the local concentration of pairing proteins at the promoter. In the absence of DNA, approximately 20–30 types of TFs directly associate with RNAP [98, 99].

Figure 2: Tetramer structure of LacI [100, 101]. (a) When the opening cleft appears, the interdimer lock cleaves [102].

5.2 Motifs

A TF-specific operator is simply a collection of nucleotides that form a motif or consensus sequence, because the interaction between a TF and DNA allows for some structural freedom. LacI has a specific motif named the helix-turn-helix (HTH) motif. This motif is found in basal and specialized TFs of all three superkingdoms. The HTH domain’s core is an open tri-helical bundle that normally binds DNA with the third helix. The tetra-helical bundle, winged helix, and ribbon-helix-helix type configurations are only a few of the structural elaborations created by the HTH domain based on the fundamental three-helical core [100].

The HTH motif and the loop that follows the recognition helix make up the major groove-binding region, while the C-terminal hinge-helix makes up the minor groove-binding region in the interactions between DNA and the TF. This interaction, often referred to as the protein–DNA interaction, was elucidated by analyses of hydrogen bonding and apolar contacts [101]. The recognition helix, which is the second helix of HTH motif, is shaped like the main DNA groove and protrudes from the protein’s surface. Shape complementarity facilitates the protein to bind to both sequence-specific and non-specific DNA locations [103].

There is a single binding site for each repressor monomer, and specific interactions stabilize the complex. The effector molecules bind to a pocket located at the junction of the repressor’s N-terminal and C-terminal subdomains. The binding site of these effector molecules is approximately 40 Å away from the operator binding site and the HTH motif [8, 100].

Each arm of LacI TF contains a core domain and a DNA-binding head group domain. The core domains are subdivided into two subdomains by lactose-binding sites. The lactose-binding sites inside the core domains are left open for lactose molecules to enter, disrupt the repressor, and induce the expression of genes in the lac operon. This architecture is necessary for the function of LacI TFs because the protein binds two operators with its two head groups and holds them close together, enforcing the interoperator loop. In addition, LacI TFs need to first span a considerable distance to find operators. Once they bind to operators, the fluctuations in the created DNA loop are readily absorbed due to the high degree of structural flexibility resulting from the loose connections between the LacI domains [76, 80, 100, 102].

Under the experimental conditions in the study of Rutkauskas et al. [94], loop formation was significantly influenced by the flexibility of the LacI protein and the opening of the tetrameric structure to a variety of angles. According to their findings, the protein can adopt different structures to accommodate various loop geometries. Under various DNA topological conditions, this structural flexibility is likely to play a significant role in looping in vivo.

The affinity of each LacI protein for operators in DNA and the amino acid sequence of LacI are not the same thing because of the molecular nature of the interactions. As a result, lower affinity is associated with an operator motif that is more degenerate. This may be a characteristic of LacI operator motifs [9, 19, 104], according to analyses of the specific operator motifs for 12 LacI family TFs in L. plantarum WCFS1 that function as specific and generic regulators [19]. The operator motif for LacI TFs contains a conserved core CG group. Structural analyses have shown that the hinge-helix residue inserts into the central CG group in the minor groove and bends the DNA. This is due to LacI’s dominance of binding coupled with an optimal binding site backbone provided by the conserved sequence (T)G-A-CG-T-C(A) [104]. Where the central "CG" sequence is the most crucial for specific binding. Although, the specific binding sequence can vary slightly based on the specific bacterial species and context [105].

6. Conclusion

The functions, mechanisms, and specifications of the LacI family are discussed in this paper. Although the majority of LacI family members are repressors of gene expression, certain members in particular bacterial species operate as activators. The role of these TFs is affected by factors that trigger transcription as well as interactions with the operator and the presence or absence an inducer. Further research is required to explore LacI binding sites and motifs in more detail. This is important for tracking the evolution of transcriptional regulatory mechanisms in bacteria.

Acknowledgments

Dina Istiqomah thanks the Japanese government (MEXT) for the award of a Monbukagakusho scholarship for studying in Japan. We thank Jennifer Smith, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

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