|Home | About | Journals | Submit | Contact Us | Français|
For many, the genetic code was to provide the key to understanding protein synthesis. For those with a bit more sophistication, it was the Shine-Dalgarno region (in prokaryotes) or scanning and the Kozak consensus sequence (in eukaryotes). In all of these, the answer was in the nucleic acid sequence of the mRNA and was based on the assumption of a single mechanism to explain translation initiation. However, like all of the macromolecular synthetic pathways, there have been two repetitive observations. First, they are considerably more complicated than anticipated, and second, there is not a single mechanism.
Beginning in the late 1980s, it was becoming apparent that there were a number of “nonstandard” events associated with translation, especially in eukaryotic systems. Much of this fell under the description of “post-translational control of gene expression,” and many of the examples would, indeed, reflect changes in the initiation efficiency of specific mRNAs. More recent examples also reflect changes in mRNA stability, alternate splicing, or microRNAs.
The intent of this minireview series is to prepare readers for the time when translation becomes a part of their research, especially for those interested in the regulation of gene expression as it relates to the basic mechanisms of processes, such as development and disease. I will initiate this minireview series with a general review of issues to be covered and will point to a number of issues that are either not like “globin mRNA” or for which there is currently no consensus. Among these issues are cap-dependent translation with long or structured 5′-untranslated regions, mRNAs with upstream open reading frames (How do they really work?), cellular IRES2 elements (Do they really exist?), cross-talk of translation with the cell, etc.
The next two articles will focus on “standard translation initiation studies” that relate to the mechanism and the use of genetics to evaluate translation in vivo. Michael Altmann and Patrick Linder will describe how the “awesome power of yeast genetics” has provided and will provide answers to the mechanisms involved in translation. To date, the mechanics of protein synthesis have been elucidated largely in vitro (and mostly using mammalian systems). However, much of our understanding of the regulation of this process has come from in vivo studies using yeast. Past examples include Thomas Donahue's work (SSL and SUI mutants), use of temperature-sensitive mutants, Alan Hinnebusch's study of the amino acid regulation of the transcription factor GCN4, and others.
Jon Lorsch and Thomas Dever offer their perspectives on the biochemistry/structural biology findings relating to translation initiation. Although many are familiar with the almost heroic efforts that have resulted in the crystal structure of the bacterial ribosome, equally impressive data on the structure of ribosome/factor complexes and the complexity of the eukaryotic initiation pathway are emerging. For the first time, structure, function, and kinetics are being combined to explain the mechanism and regulation of protein-synthesis initiation.
Most of the current in vivo studies use yeast or tissue culture cells. However, what does one do if one wants to consider a real, intact multicellular organism? What type of signals predominate, why is leucine the “magic amino acid,” and just how many signaling pathways might be at work to confound the investigator? The key feature is that multicellular organisms, especially mammalian systems, maintain homeostasis. Thus, attempts to isolate effects to specific organs have been challenging. Scot Kimball and Leonard Jefferson will provide us with insights into where new methodologies are needed to relate effects on protein synthesis to the overall goal/strategy of the organism for survival.
The study of IRESs began with the groundbreaking work of Nahum Sonenberg, Eckard Wimmer, and their colleagues that examined the initiation of poliovirus and encephalomyocarditis virus mRNAs. Subsequent studies, mostly using mammalian systems, have examined the existence of cellular IRES elements that are present in normal cellular mRNAs. In stark contrast to progress in the characterization of viral IRESs, which has produced detailed molecular and even structural descriptions of several distinct mechanisms, the work with cellular IRES elements has been more refractory because they are generally inefficient. Wendy Gilbert will describe current studies in this area as well as her own efforts to identify and characterize IRES elements in yeast. The real task is to define the mRNAs with IRES elements and to begin to explore their regulation (IRES trans-acting factors anyone?) in vivo, and perhaps, sooner or later, in vitro.
As is true for any key macromolecular pathway, protein synthesis seems to be a thermometer for cellular good health. A variety of signals trigger the alteration of protein synthesis, either as the absolute level of expression (i.e. as total amino acid incorporation) or as the relative alteration of expression of the population of mRNAs within the cell (as competition or as specific activation or repression of existing mRNAs). A commonly identified protein associated with such changes is eEF1A, the most abundant of the translation factors at about 2% of the total cellular protein. Maria Mateyak and Terri Goss Kinzy will describe many of their studies that link eEF1A function with cytoskeletal formation and more recent research that implicates this protein in regulating the interaction of the ribosome and the cell. Identifying the proteins that interact with eEF1A may explain concepts of localized translation and the influence of changes in cellular morphology on translation at different stages of the cell cycle.
One of the potentially important new areas of research involves the role of small noncoding RNAs in cell growth and development and their use to manipulate the levels of gene expression in systems that do not have the genetic plasticity found in yeast. Although both small interfering RNAs and microRNAs are now part of the molecular biologist's toolbox, microRNAs have been associated with a variety of processes. An example is the recent work of Joan Steitz and her colleagues showing that microRNAs are either positive or negative regulators of gene expression. Timothy Nilsen will explore what has been learned about these RNAs and whether we might expect that there is a common mechanism for both or whether there might be a series of unique regulatory pathways.
In an intriguing new area of research, progress is being made in determining the role of protein synthesis in learning. Previous efforts using drugs have shown that unlike short term memory, long term memory requires protein synthesis. How does this work? With the recent advances in understanding the mechanism of protein synthesis and the characterization of the proteins/factors that are required, it is now possible to examine what proteins are involved in this process. Nahum Sonenberg, Christos Gkogkas, and Mauro Costa-Mattioli will review our current knowledge and some suggestions as to where research is going in terms of examining learning at the molecular level. How long will it be before we have a real mechanistic interpretation, and how might we get there? The GCN2−/− mouse is a start. What is next?
Finally, we will present a recap of this year's Nobel Prize awards for the structure of the ribosome. Although much of the early work from the laboratories of Ada Yonath and Thomas Steitz focused on the structure of the bacterial ribosome in the absence of protein synthesis components, the Venki Ramakrishnan group provided mechanistic insights into the catalytic workings on the surface of the bacterial ribosome. One of the participants in this Nobel effort, Dr. Poul Nissen, and his colleague Charlotte Knudsen, will provide us with an update of the current workings of the ribosome as it relates to the molecular enzymology of protein biosynthesis.
The variety of events that affect the regulation of translation, specifically or globally, and the site-specific translation of proteins (as best evidenced in neurons) have led to exceptionally diverse patterns of expression of proteins. These, coupled with the already known controls at the level of transcription, mRNA processing, and mRNA turnover (at the level of macromolecular events) and the post-translational modifications that alter enzyme/factor activities, ensure that multicellular organisms have many mechanisms to control growth and development and to maintain homeostasis.
*This minireview will be reprinted in the 2010 Minireview Compendium, which will be available in January, 2011.
2The abbreviation used is: