Two lines of in vivo evidence suggest that the loop between araI and araO2 is broken upon the addition of arabinose. First, the operon is fully inducible if araO2 is deleted. This implies that the loop is not involved with induction. Second, the occupancy of araO2 falls immediately after arabinose addition. One good way to study such regulated looping is to perform in vitro experiments. AraC loops sufficiently weakly, however, that looping of linear DNA just does not occur. Instead, looping had to be studied using small supercoiled circles of about 400 base pairs. These are sufficiently small, and the supercoiled DNA is sufficiently wound upon itself, that AraC protein can easily bridge the gap between araO2 and araI. These supercoiled, looped circles migrate upon electrophoresis at a rate different from free circles or circles with AraC bound at a single point. Therefore binding, looping, and unlooping can all be assayed.

Study of the looped supercoils revealed an unexpected property. As AraC is a dimer in solution, and a dimer binds to linear DNA containing the araI site, it had seemed likely that the looped species would be formed from a dimer of AraC bound to araI and a dimer bound to araO2. This proved not to be the case. The looped species contains only one dimer! In the absence of arabinose one of the monomers of an AraC dimer binds to the left half of araI, and the other monomer binds to araO2 (Fig.1). The left and right halves of araI are called araI1 and araI2. Upon the addition of arabinose the protein reorients, and the subunit contacting araO2 lets go, and contacts araI2. The subunit rearrangement occurs in the absence of free protein and is largely independent of the precise sequences at the sites involved. In the absence of arabinose the protein prefers to loop, that is, contact to nonlocal sites. In the presence of arabinose, the protein prefers to contact local sites. The contacting of the araI2 site by AraC protein provides the inducing signal to RNA polymerase. Only if this site is properly positioned so as  to overlap partially the -35 sequence of P_BAD does AraC activate transcription.

Fig1. Reorientation of the two subunits of AraC protein shifts the most favored conformation from a looped state to the unlooped state.

One simple mechanism that could generate the observed behavior of AraC protein is a subunit reorientation. Suppose that in the absence of arabinose the subunits are oriented such that binding to two half-sites in a looping structure is energetically easier than binding to two adjacent half-sites. Then the protein would prefer to loop in the absence of arabinose. If the presence of arabinose causes the subunits to reorient so that binding to the two half-sites of araI is favored, then the protein would unloop, bind to both halves of araI, and induce P_BAD .

The data supports a slight modification of the reorientation model outlined above. AraC protein monomers consist of a dimerization domain flexibly connected to a DNA-binding domain. The half-sites to which AraC binds normally are oriented in a direct repeat orientation. AraC still binds with high affinity when one half-site is inverted so that the total site possesses inverted repeat symmetry. This shows that the dimerization and DNA-binding domains are flexibly connected. Fur ther, the half-sites of araI can be separated by an additional 10 or 21 base pairs, or even inverted, without greatly affecting the affinity of AraC. When, however, arabinose is added, the affinity of AraC for the wild-type araI site, the “+10” site, and the inverted site is increased, but that for the “+21” site is decreased. Because arabinose did not increase the affinity of AraC for all the sites, arabinose cannot be increasing the intrinsic affinity of a DNA-binding domain for a half-site. Instead, arabinose alters the relative positions of the DNA-binding domains or changes the flexibility or length of the connection between the dimerization and DNA-binding domains (Fig. 2).

Fig2. Binding of AraC protein to sequences with different distances separating the half-sites or with one half-site inverted. In the presence of arabinose, the reach of AraC is reduced.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم الإعلام يشارك في الاجتماع التحضيري لمناقشة الخطة الإعلامية لزيارة عاشوراء

بحضور متوليها الشرعي.. العتبة العباسية المقدسة تواصل إقامة المجالس الحسينية لإحياء شهر المحرم

قسم الشؤون الفكرية يطلق برنامجه التبليغي في أكثر من 20 دولة إفريقية

شعبتا الخطابة النسوية والتوجيه الديني تواصلان إقامة المجالس الحسينية في مرقد أبي الفضل العباس (عليه السلام)

المزيد

الآخبار الصحية

دراسة تكشف علاقة خطيرة بين عمر الأم وصحة مولودها

دراسة تحذر.. اضطراب نوم بسيط قد يسبب الموت المبكر ؟

الاستحمام بالماء الساخن.. راحة مؤقتة وأضرار دائمة للبشرة

ماذا يحدث للجسم عند تخطي وجبة الإفطار؟

المزيد

الآخبار التكنلوجية

كيف تساعد الصيانة الوقائية في الحفاظ على كفاءة التكييف في سيارتك ؟

في ظاهرة غريبة حيرت العلماء.. كوكبنا قد يسجل أقصر يوم في التاريخ هذا الصيف!

السد الصيني العملاق يعيق دوران الأرض ويغيّر شكلها ؟

السعودية تطلق 10 تجارب علمية إلى محطة الفضاء الدولية

المزيد

مواضيع ذات صلة

DNA Looping and Repression of araBAD

Binding Sites of the ara Regulatory Proteins

Endoplasmic Reticulum-Associated Degradation and the Unfolded Protein Response

Protein Folding and Modifications in the Endoplasmic Reticulum

Protein Modifications

Regulating AraC Synthesis

Repression by AraC

Detection and Isolation of AraC Protein

Genetics of the Arabinose System

The Eukaryotic Genome

Nucleic Acids and Their Organization in Eukaryotic, Prokaryotic, and Viral Genomes

The Sugar Arabinose and Arabinose Metabolism