Tuberculosis (TB), caused by the bacteria Mycobacterium tuberculosis (Mtb), is one of the world’s deadliest infectious diseases. One mechanism by which Mtb can develop resistance to current TB treatments and adapt to stress conditions in a host is through regulation of gene expression. Translation efficiency can impact gene expression and is itself influenced by characteristics of a gene’s ribosomal binding site (RBS) within the 5’ UTR. Here we used both computational and experimental approaches to investigate RBS characteristics to determine their impact on translation efficiency in the model organism Mycolicibacterium smegmatis. We focused on the presence of Shine-Dalgarno (SD) sequence motifs and secondary structure. Sequences complementary to the loops of the B11 sRNA, which is thought to play a role in the regulation of translation efficiency in mycobacteria, were inserted in the RBS of fluorescent reporters. The insertion of these sequences decreased fluorescence in both wild-type and ΔB11 strains, but for one of them the decrease was greater in the wild-type strain, consistent with the idea that B11 negatively regulates translation. We then analyzed the relationship between SD motifs and ribosome occupancy. When controlling for secondary structure we found that the presence of any SD motif with 4 nucleotides or more of complementarity to the anti-SD was associated with higher ribosome occupancy. However, there were not differences in ribosome occupancy among genes with SD motifs of different lengths or with complementarity to different parts of the anti-SD. We also assessed the impact of SD motif location on ribosome occupancy. We found that there seems to be an optimal position for the SD motif relative to the start codon that is not greatly affected by which part of the anti-SD the SD binds to. Our results may be used in further investigation of the role of B11 and in future plasmid designs to maximize gene expression.

• This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

• Masiello, Catherine

Publisher

• Worcester Polytechnic Institute

Identifier

• E-project-042922-212002
• 66401

Keyword

• Ribosome Binding Site
• M. smegmatis
• Translation
• Shine-Dalgarno
• Mycobacteria

Advisor

• Shell, Scarlet

Year

• 2022

Date created

• 2022-04-29

Resource type

• Major Qualifying Project

Major

• Biology & Biotechnology

Rights statement

• In Copyright