RNAs have a wide range of functions as messengers, amino acid adaptors, ribozymes, and regulators, and play fundamental roles in all organisms. Escherichia coli (E. coli), an extensively studied model organism in the prokaryotic domain, has contributed significantly to our understanding of the versatile functions innate to this class of molecules. This PhD thesis focuses on the transfer RNAs (tRNAs) and small regulatory RNAs (sRNAs) of E. coli. While both RNA classes encompass relatively short RNA molecules, the two classes differ greatly in their biological role and in the heterogeneity of the RNA species within the classes. Further, while the tRNAs constitutes a well-defined group of molecules, the field of sRNAs is rapidly evolving with more species, mechanisms and phenotypes being assigned continuously.

In the presented work, we investigated the regulatory patterns of tRNAs and sRNAs in the context of amino acid starvation. Our findings revealed a contrasting response between these two RNA classes. While tRNAs exhibited remarkable stability, even under amino acid-deprived conditions, sRNAs displayed a wide range of regulated behaviors. These results underline a difference between the relatively uniform tRNA class compared to the highly heterogeneous sRNA class.

Another distinguishing factor between these two RNA classes lies in how well the classes are defined. While it is generally presumed that all tRNA species in E. coli have been identified, the presence of many undefined sRNA genes in the same organism is widely acknowledged.

To contribute to closing this knowledge gap within the sRNA field, we set out to identify novel putative sRNA genes. We developed two methods for the purpose and validated the expression of a novel putative sRNA identified by both methods. The putative novel sRNA exhibited differential expression in response to amino acid starvation, which could hint a function within stress response regulation.

RNA modifications have classically been reserved for the ribosomal RNA (rRNA) and tRNA classes in prokaryotes. However, emerging evidence points to 5’ RNA capping as a prevalent modification found on prokaryotic messenger RNAs (mRNAs) and sRNAs. We employed the

CapZyme-Seq method to identify capped RNAs in E. coli. Intriguingly, among the capped RNA species detected in our study, we identified several sRNAs and mRNAs, which have not previously been reported to be capped. Among these, we validated high capping levels of the sRNA CyaR suggesting CyaR as a promising candidate for further exploration of the unknown mechanisms and functions associated with 5’ RNA capping in prokaryotes.

Additionally, we explored potential biases while working with RNA in the laboratory. We identified alarming biases in commonly used RNA extraction methods when using them for extracting RNA from stressed E. coli cultures. Further, we recognized the importance of taking RNA modifications into consideration when designing oligo probes for northern blotting. Acknowledging such biases is crucial in ensuring an accurate and reliable analysis of RNA levels.

Conclusively, this thesis advances our knowledge within the field of untranslated RNAs of E. coli by developing methodologies for identification of putative novel sRNA species, by suggesting a model sRNA candidate for future studies of 5’ RNA capping, by exploring the regulation of untranslated RNAs in response to amino acid starvation, and by emphasizing the importance of recognizing and eliminating biases in established experimental approaches.