Background:In yeast, eIF4A and eIF4F are essential for efficient translation initiation, but our understanding of their mechanisms of action is rudimentary. Results: A detailed biochemical analysis of yeast eIF4A, alone and within eIF4F, is described. Conclusion: The eIF4G subunit confers specificity for unwinding duplexes with 5Ј-overhangs on yeast eIF4F. Significance: The observed 5Ј-end specificity may play a role in ribosomal scanning along mRNA.

In Escherichia coli, the cold shock response occurs when there is a temperature downshift from 37°C to 15°C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3-to-5 processing exoribonucleases of E. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed that in vivo deletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activity in vitro independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases in E. coli.When exponentially growing cells of Escherichia coli are shifted from 37°C to a low temperature, such as 15°C, a cold shock response is elicited. This response is characterized by a transient arrest of cell growth termed the acclimation phase, followed by resumption of growth at the low temperature. During the acclimation phase there is severe inhibition of general protein synthesis. However, several cold shock proteins are induced during this phase, including CspA (19) and its homologues, such as CspB (26) CsdA is a DEAD-box protein that belongs to the large family of putative RNA helicases. Members of this family are conserved in organisms from bacteria to humans (29) and play important roles in many cellular processes, such as processing, transport, or degradation of RNA or ribosome biogenesis (for a review, see reference 21). CsdA has been identified as a multifunctional protein, and it has been proposed that this protein participates in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. CsdA is essential at low temperatures, and deletion of its gene impairs growth when there is a cold shock (8, 24). On the other hand, it is dispensable at 37°C. Previously, we showed that the helicase activity of CsdA is pivotal in its role at low temperature (3). Our in vivo genetic screening of an E. coli strain revealed that another DEAD-box RNA helicase, RhlE...

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.Helicases are proteins that translocate in one specific direction along the nucleic acid backbone to catalyze processes such as the separation of the complementary strands of a double helical nucleic acid, recombination of DNA molecules, or remodeling of proteins bound to nucleic acids (1-5). Helicases are referred to as motor enzymes, because they convert the chemical energy of NTP hydrolysis (a reaction with a negative ⌬G) into mechanical force for motion and strand separation. It is not clear whether the force they generate is used predominantly to translocate on the nucleic acid or whether it is directed mainly at actively destabilizing and melting the double-stranded nucleic acid. It is also not known whether all helicases share a common mechanism or whether they act via different strategies to catalyze strand separation. Therefore, it is difficult to predict a priori whether and how the helicase activity would depend on nucleic acid base pair stability.Helicases encounter a range of nucleic acid sequences as they translocate on and unwind the strands of double-stranded DNA or RNA in the cell. It is well known that the base sequence dictates the stability of the double helical nucleic acid. The GC base pair in nucleic acids is more stable than the AT base pair. Similarly, the neighboring base sequences and the stacking energies between adjacent base pairs determine the overall stability of the double helical nucleic acid (6). A strong dependence of helicase activity on base pair stability would adversely affect controlled biological processes, such as the coordination of leading and lagging strand DNA synthesis during genome replication or the repair and recombination of specific DNA sequences. On the other hand, the dependence of helicase activity on base pair stability pr...