Initiation of translation is regulated by the accessibility of ribosomes to the Shine-Dalgarno sequence. This stretch of four to nine purine residues are located upstream the initiation codon and hybridize to a pyrimidine-rich sequence near the 3 end of the 16S RNA within the 30S bacterial ribosomal subunit. Polymorphism in this particular sequence has both positive and negative effects on the efficiency of base-pairing and subsequent protein expression. Initiation is also regulated by proteins known as initiation factors which provide kinetic assistance to the binding between the initiation codon and tRNA, which supplies the 3-UAC-5' anticodon. IF1 binds the 30S subunit first, instigating a conformational change that allows for the additional binding of IF2 and IF3. IF2 ensures that tRNA remains in the correct position while IF3 proofreads initiation codon base-pairing to prevent non-canonical initiation at codons such as AUU and AUC. Generally, these initiation factors are expressed in equal proportion to ribosomes, however experiments using cold-shock conditions have shown to create stoichiometric imbalances between these translational machinery. In this case, two to three fold changes in expression of initiation factors coincide with increased favorability towards translation of specific cold-shock mRNAs.