DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. E nergy production in eukaryotic cells depends upon the function of mitochondria, double-membrane organelles that are thought to have evolved from an ancient bacterial endosymbiont (1, 2). While most proteins that are essential for mitochondrial (mt) function are encoded by nuclear genes and imported into the organelle, a subset of critical proteins, mostly components of the oxidative phosphorylation complex and mitochondrion-specific ribosomal and tRNAs, are encoded in the mt genome (3, 4). Therefore, expression of nuclear and mt genes must be coordinately regulated (5, 6). As a consequence, mitochondria have developed a highly active RNA surveillance/degradation system to ensure the integrity of mt transcripts and to regulate the availability of mt mRNA by balancing the rates of RNA synthesis and degradation (7,8,9).Little is known about how transcription is controlled in mitochondria and what proteins participate in adjusting the rates of mt RNA synthesis and degradation. In the yeast Saccharomyces cerevisiae, mt transcription is carried out by an RNA polymerase (RNAP) consisting of a single-subunit catalytic core (Rpo41p) that is related to T7 RNA polymerase (T7 RNAP) and an additional transcription factor (Mtf1p) for initiation (10,11,12). RNA degradation in turn depends upon a two-subunit mt degradosome that includes Suv3p, a processive helicase that unwinds double-stranded RNA (dsRNA), and Dss1p, a 3= to 5= exonuclease (13,14). Importantly, deletion of SUV3 is suppressed by mutations in RPO41 and MTF1, suggesting that alterations in these components of the transcription system help to restore the balance between RNA synthesis and degradation (8). The SUV3 deletion is also suppressed by overexpression of Mss116p, an mt DEAD box protein that is involved in RNA splicing, processing, and possibly translation (15,16). While this effect might be attributed to compensation for Suv3p deficiency by a protein that has RNA unwinding activity, we have recently found that Mss116p is associated with the mt trans...