Sequences and proposed structures of native and mutated 5′-UTRs.

<p>(A) Nucleotide sequences. A putative stem loop of the native <i>hp</i> 5′-UTR was changed by <i>in vitro</i> mutagenesis. The mutated nucleotides are shown in grey and are underlined. (B) <i>In silico</i> structural analysis of the stabilized, the native, and the destabilized 5′-UTRs. The structural analysis were performed by using the mfold 3.2 program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004484#pone.0004484-Mathews1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004484#pone.0004484-Zuker1" target="_blank">[41]</a>. Mutated nucleotides are indicated by a bar. The start codon of the orf is also included and marked by an arrow.</p

The structure of the 5′-UTR can influence translational efficiency and translational regulation.

<p>Specific putative structure elements were stabilized and destabilized in a native 5′-UTR (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004484#pone-0004484-g003" target="_blank">Figure 3</a>). Translational efficiencies of the mutated versions and the wild type were determined by using the reporter gene system. (A) The transcript fusions are shown schematically. The DHFR enzymatic activities, the <i>dhfr</i> transcript levels and the translational efficiencies of exponential and stationary growth phases are tabulated. Three biological replicates were performed and average values with standard deviations (in parenthesis) were calculated. (B) The translational efficiencies after normalization to the control transcript without UTRs (No. 1) are shown.</p

The direction of translational regulation is determined by the 3′-UTRs.

<p>Using the reporter gene system different combinations of 5′- and 3′-UTRs of the <i>hlr</i> and the <i>hp</i> transcript were studied. (A) The transcript fusions are shown schematically. The DHFR enzymatic activities, the <i>dhfr</i> transcript levels and the translational efficiencies of exponential and stationary growth phases are tabulated. Three biological replicates were performed and average values with standard deviations (in parenthesis) were calculated. (B) The translational efficiencies after normalization to the control transcript without UTRs (No. 1) are shown.</p

Plasmids and characteristic features.

<p>Plasmids and characteristic features.</p

Two differentially translated <i>H. volcanii</i> genes and their UTRs.

<p>(A) The lengths of the 5′- and 3′-UTRs of the <i>hp</i> and the <i>hlr</i> transcripts are tabulated. The UTRs of the genes were determined in a prior study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004484#pone.0004484-Brenneis1" target="_blank">[22]</a>. (B) The sequences of the 5′- and 3′-UTRs of the <i>hlr</i> and the <i>hp</i> transcripts are shown. The start as well as the stop codon of the orf are also included and printed in bold. (C) Growth phase-dependent differential translational efficiencies of the <i>hlr</i> and <i>hp</i> genes. The values were obtained by isolating free, non-translated RNAs as well as polysome-bound RNAs and their genome-wide comparison with DNA microarrays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004484#pone.0004484-Lange1" target="_blank">[7]</a>. The results were normalized to the average of all genes. The ratio of polysomal to free RNA for the <i>hlr</i> and <i>hp</i> transcripts are shown for exponentially growing and stationary phase cells.</p