Initiation complex prokaryotic

C. Eukaryotic transcription is more complex than in prokaryotes, mainly in terms of the nature of the RNA polymerases, the assembly of the pre-initiation complex, and the need for processing eukaryotic RNAs. 

Initiation of protein synthesis involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP (which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex (see Figure 31.13). [Note In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukary- otes, there are at least ten (designated elF to indicate eukaryotic origin).] There are two mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation ... 

Formation of the initiation complex for protein synthesis in prokaryotes. E. coli has three initiation factors bound to a pool of 30S ribosomal subunits. One of these factors, IF-3, holds the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors, IF-1 and IF-2, promote the binding of both fMet-tRNAfMel and mRNA to the 30S subunit. The binding of mRNA occurs so that its Shine-Dalgamo sequence pairs with 16S... 

Formation of the initiation complex for protein synthesis in eukaryotes. The reaction begins with the small subunit held apart from the large subunit by an antiassociation factor and ends with the hydrolysis of GTP and joining of the large subunit as in prokaryotes. The intervening reactions are different. A much more complex spectrum of initiation factors (elFs) is involved, and the exact function of only a few of these factors is known with certainty. The... 

Initiation in prokaryotes involves binding of mRNA by small ribosomal subunit (30S), followed by association of the fMet-tRNAmet (initiator formyl-methionyl-tRNAmel) that recognizes the initiation codon. Large ribosomal subunit (50S) then joins to form the 70S initiation complex. In eukaryotes, the initiator aminoacyl-tRNA is not formylated. Instead Met-tRNAf161 forms 40S preinitiation complex with small ribosomal subunit (40S) in the absence of mRNA. The association of mRNA results in a 40S preinitiation complex, which forms an 80S initiation complex after large ribosomal subunit (60S) joins. 

One of the first metalloregulatory proteins to be characterized extensively is the prokaryotic MerR transcription factor (1, 6, 7), which acts either as a repressor (apo-protein) or an activator (holo-protein) of the mer operon encoding mercury resistance proteins (Fig. Ic). The —35 and —10 sequence elements of the mer promoter, binding sites for the RNA polymerase initiation complex, are separated by an unusually long distance that results in poor constitutive transcription. Apo-MerR binds to the DNA between these sequences and bends the DNA, which results in a slight increase in repression on the suboptimal promoter. It also recruits the RNA polymerase to the transcription start site where it waits in a stalled complex. Upon binding of... 

Figure 29.27. Translation Initiation in Prokaryotes. Initiation factors aid the assembly first of the 308 initiation complex and then of the 70S initiation complex.

Elongation and termination. Eukaryotic elongation factors EFla and EFip y are the counterparts of prokaryotic EF-Tu and EF-Ts. The GTP form of EFla delivers aminoacyl-tRNA to the A site of the ribosome, and EFip y catalyzes the exchange of GTP for bound GDP. Eukaryotic EF2 mediates GTP-driven translocation in much the same way as does prokaryotic EF-G. Termination in eukaryotes is carried out by a single release factor, eRFl, compared with two in prokaryotes. Finally, eIF3, like its prokaryotic counterpart IF3, prevents the reassociation of ribosomal subunits in the absence of an initiation complex. 

Protein synthesis takes place in three phases initiation, elongation, and termination. In prokaryotes, mRNA, formylmethionyl-tRNAf (the special initiator tRNA that recognizes AUG), and a 308 ribosomal suhunit come together with the assistance of initiation factors to form a 308 initiation complex. A 508 rihosomal suhunit then joins this complex to form a 708 initiation complex, in which fMet-tRNAf occupies the P site of the rihosome. 

D. Streptomycin causes misreading of mRNA codons and, thereby, prevents formation of the initiation complex in prokaryotes. 

Early steps in protein synthesis in prokaryotes formation of the 30S preinitiation complex and 70S initiation complex. 

Assembly of the RNA pol II initiation complex in eukaryotes requires a much larger number of proteins than what is needed to form a transcriptional initiation complex in prokaryotes. What answer best explains the reason for this difference ... 

Only tRNAf is accepted to form the initiation complex. All further charged tRNAs require fully assembled (i.e., VOS) ribosomes. All prokaryotic proteins are synthesized with the same N-terminal residue, N-formylmethionine. 

The initiation process differs for prokaryotes and eukaryotes (Table 15.3). In bacteria, the initiating methionyl-tRNA is formylated, producing a formyl-methionyl-tRNAf that participates in formation of the initiation complex (Fig. 15.9). Only three initiation factors (IFs) are required to generate this complex in prokaryotes, compared with the dozen or more required by eukaryotes. The ribosomes also differ in size. Prokaryotes have 70S ribosomes, composed of 308 and 50S subunits, and eukaryotes have SOS ribosomes, composed of 40S and 60S subunits. Unlike eukaryotic mRNA, bacterial mRNA is not capped. Identification of the initiating AUG triplet in prokaryotes occurs when a sequence in the mRNA (known as the Shine-Dalgamo sequence) binds to a complementary sequence near the 3 -end of the 16S rRNA of the small ribosomal subunit. 

The 3 phosphorothioate residues, introduced by the phosphoramidite method, were shown to significantly protect the oligomer from the action of nucleases. This hybrid, in which the ribonucleotide portion is complementary to the leader sequence of phage fl coat protein mRNA, was used to study the formation of the initiation complex in prokaryotic translation. 

Once associated with mRNA, the pre-initiation complex scans downstream to locate a start codon (AUG). This process is driven by ATP, and requires the helicase activity of an elF. The start codon is recognised by base paring between the anti-codon on tRNA and the AUG on the mRNA. Codon-anticodon interaction is facilitated by yet more elFs. Usually, the first AUG codon that is encountered is used, especially if it is surrounded by the so called Kozak consensus sequence (named after its discoverer Marilyn Kozak). Occasionally a later AUG is used if the first AUG not in the context of a consensus Kozak sequence, or is very close to the 5 cap. This is in contrast to prokaryotes, where the AUG that will act as a start codon is located at the future P site by the position of the ribosome after base-pairing between the 16S rRNA of the ribosome and the Shine Dalgamo sequence. 

The selection of the proper initiation codon is aided by the base pairing between a pyrimidine-rich sequence at the 3 -end of 168 rRNA and complanentary pnrine-rich tracts of 3-lOnt (known as 8hine-Dalgamo sequence) at the 5 -end (centering 10nt upstream from the initiation codon) of prokaryotic mRNA. The assembly of the initiation complex and initiation of synthesis also require the participation of various soluble initiation factors (IPs, Table 13.8) in the initiation events ... 

Fig. 2. Formation of a stable initiation complex between a 70 S ribosome and messenger RNA. In the final complex fMet-tRNAf " is in the correct position for the formation of a peptide bond. IF-1, IF-2, and IF-3 are the protein initiation factors and fMet-tRNAf " is the formyl methionyl tRNA which is used for the initiation of protein synthesis in prokaryotes. The process in animal cells is thought to be substantially the same, the initiation factors being termed IF-Ml, IF-M2, and IF-M3 and the initiator tRNA, Met-tRNAt . The methionine attached to this tRNA species is not normally formylated but can be so modified by enzymes from bacterial cells.

In prokaryotes and eukaryotes, the initiation complex is prepared for the addition of the large ribosomal subunit by the release of initiation factor 3. In bacteria, the 508 subunit appears simply to replace IF-3, while IF-1 and IF-2 leave the complex afterward. In eukaryotes, another factor, eIF-5, catalyses the departure of the previous initiation factors and the joining of the 608 subunit. In both cases, release of initiation factor 2 involves hydrolysis of the GTP bound to it. Also, the Met-tRNA, is bound to the P site of the large ribosomal subunit. 

Diagrammatic representation of translation on prokaryotic ribosomes. The elongation cycle starts by interaction of the 70S initiation complex with fMet- tRNA EFTu GTP. In all subsequent rounds of the cycle, fMet-tRNArEFT tGTP interacts with the mRNA ribosome complex carrying the growing polypeptide chain. Termination occurs when n amino acids have been incorporated, where n represents the number of codons between the initiation codon AUG and the termination codon (in this example UAA). 

The ribosome cycle starts with the stepwise formation of an initiation complex from mRNA, charged initiator tRNA, and ribosomal subunits. A number of pre-initiation complexes are formed as intermediates and the process is facilitated by initiation factors. In outline, prokaryotic and eukaryotic systems are similar, but there are a few differences, particularly as regards the complexity of the initiation factors and details of the mechanisms. 

Zagorska, L., Chroboczek, J., Klita, S., and Szafranski, P., 1982, Effect of secondary structure of mRNA on the formation of initiation complexes with prokaryotic and eukaryotic ribosomes, Eur. J. Biochem. 122 265. 

FIGURE 5 Translation initiation. In prokaryotes, three initiation factors are responsible for assembling the initiation complex prior to decoding of a message. The mRNA and initiator tRNA bind to the small ribosomal subunit in random order, with IF2 selectively binding initiator tRNA. Hydrolysis of IF2-bound GTP promotes formation of the SOS initiation complex. Initiation Factors 1 and 3 leave the complex, and the large ribosomal subunit binds to form the 70S initiation complex with release of IF2. 

Many more protein factors are involved in eukaryotic initiation some systems contain more than 10 initiation factors. Particular features of translation initiation are also different in the higher organisms. Most notably, prokaryotic ribosomes can initiate internally on an mRNA (even on circular RNAs), while in eukaryotes a preinitiation complex binds to the 5 -end of the mRNA and then progresses to an initiation complex. Eukaryotic mRNAs are capped at their 5 -end with a 7-methylguanosine triphosphate structure, and one of the eukaryotic initiation factors binds this capped end. The preinitiation complex then moves along the mRNA and initiates translation at the first AUG codon it comes to. Consistent with this scanning mechanism is the observation that eukaryotic mRNAs do not contain Shine-Dalgarno-like sequences. 

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