Ribosome tRNA binding

In the cytoplasm, the mRNA attaches to a ribosome and acts as a template for the construction of a protein with the proper amino acid sequence (a process known as translation ). Single amino acids are brought to the ribosome by transfer RNA molecules (tRNA) and added to the growing amino acid chain in the order instructed by the mRNA. Each time a nucleotide is added to the growing RNA strand, one molecule of ATP is broken down to ADP. Each time a tRNA binds an amino acid and each time the amino acid is added to the protein, additional ATP is broken down to ADP. Because proteins can contain many hundreds of amino acids, the cell must expend the energy in 1,000 or more ATP molecules to build each protein molecule. 

Of the fonr possible optical isomers of chloramphenicol, only the o-threo form is active. This antibiotic selectively inhibits protein synthesis in bacterial ribosomes by binding to the 50S subunit in the region of the A site involving the 23 S rRNA. The normal binding of the aminoacyl-tRNA in the A site is affected by chloramphenicol in such a... 

Hinnebusch, A. (2000). Mechanism and Regulation of Initiator Methionyl-tRNA Binding to Ribosomes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

Hinnebusch, A. G. (2000). Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. In Translational Control of Gene Expression (N. Sonenberg,... 

An example of the results that can be obtained using the microtiter format is shown in Fig. 12.5A, which illustrates the inhibition of fMet-tRNA binding to 022 mRNA-programmed 30S ribosomal subunits caused by increasing concentrations of GE81112, the inhibitor of 30SIC formation characterized in Brandi et al. (2006b). 

Kinetics of fMet-tRNA binding to 30S ribosomal subunit Inhibition of ribosomal binding of fMet-tRNA by an antibiotic may reduce the level of initiation complex formed at equilibrium. However, if the effect of the inhibitor consists mainly of slowing down the binding reaction, its effect may appear less dramatic after a relatively long incubation time. For this... 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

Figure 7 The direct and indirect pathways of tRNA asparaginylation. The direct pathway consists of charging by AsnRS on tRNA " of free Asn formed with asparagine synthetase A or B. The Asn-tRNA " binds the EF-Tu factor in bacteria (or EF-1A in eukaryotes and archaea) to be carried to the ribosome, in the indirect pathway, a nondiscriminating AspRS (ND-AspRS) charges Asp on tRNA " Asp-tRNA " does not bind the eiongation factor but is converted by the tRNA-dependent trimeric amidotransferase GatCAB into Asn-tRNA ", which binds the EF-Tu factor and is carried to the ribosome where it is used for polypeptide chain elongation.

Amino add activation. Before binding to the ribosomes, tRNAs are loaded with the correct amino acids by specific ligases (7 see p. 248). It is the amino acid tRNA ligases that carry out the transfer (translation) of the genetic information from the nucleic acid level to the protein level. 

In the 70 S initiation complex, formylme-thionine tRNA is initially located at a binding site known as the peptidyl site (P). A second binding site, the acceptor site (A), is not yet occupied during this phase of translation. Sometimes, a third tRNA binding site is defined as an exit site (E), from which uncharged tRNAs leave the ribosome again (see p. 252 not shown). 

Occasionally, a negative activation energy is reported. For example, an value of -13.0 kJ mol was obtained for tRNA binding to the E-site of poly(U)pro-grammed ribosomes containing unlabeled tRNA at its P-site. In such circumstances, one must be especially wary, because there are several reasons why an erroneously negative E value is obtained ... 

Fig. 4 Schematic section through the small ribosomal subunit of yeast (gray) exposing the decoding region. The model is based on structural work on the prokaryotic small ribosomal subunit. The homologous proteins of the E. coli decoding region are S4, S5, and S12 (Ogle et al. 2003). Movements within the small ribosomal subunit upon cognate tRNA binding (domain closure) are denoted by red arrows. Mutations in...

Prevents premature binding of tRNAs to A site Facilitates binding of fMet-tRNA 61 to 30S ribosomal subunit Binds to 30S subunit prevents premature association of 50S subunit enhances specificity of P site for fMet-tRNA 61... 

Initiation (Figs. 29-10 and 29-11), elongation (Fig. 29-12), and termination are three distinct steps in the synthesis of a protein. A variety of specialized proteins are required for each stage of synthesis. Their sequential interaction with ribosomes can be viewed as a means of ensuring an orderly sequence of steps in the synthesis cycle. The rate of protein formation will depend upon the concentrations of amino acids, tRNAs, protein factors, numbers of ribosomes, and kinetic constants. The formation of specific proteins can also be inhibited by translational repressors, proteins that compete with ribosomes for binding to target mRNAs.287... 

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