Optimized bacterial production of nonglycosylated human transferrin and its half-molecules.

Transferrin is a glycoprotein functioning in iron transport in higher eukaryotes, and consists of two highly homologous domains. To study the function of the glycan residues attached exclusively to the C-terminal domain, we have constructed a plasmid allowing production of nonglycosylated human transferrin in Escherichia coli. By molecular biological and genetic techniques, production was stepped up to 60 mg/l.

CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli.

To activate expression of a human transferrin (Tf)-encoding cDNA in Escherichia coli by translational coupling, it was placed in an expression plasmid downstream from a 5'-terminal fragment from the replicase (R)-encoding gene of bacteriophage MS2. The resulting construct was found to produce, besides the desired Tf, a protein with the mobility of a fusion product (RTf) of the N-terminal R fragment and Tf. Analysis of available mutants showed that this fusion results from +1 ribosomal frameshifting at the end of the R reading frame.

[Kasugamycin methylase modified by ribosomal RNA from group A streptococci].

Kasugamycin sensitivity in Escherichia coli depends on the specific enzyme methylating rRNA. Native group A streptococci (GAS) were found to be sensitive to kasugamycin. After introduction of the erythromycin gene located on the transposon Tn916E into GAS some of the strains obtained kasugamycin resistance together with erythromycin resistance (erm).

Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.

In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity.

Is there a special function for U.G basepairs in ribosomal RNA?

U.G basepairs are well-established elements of RNA structure. The geometry of this pair is different, however, from classical Watson-Crick basepairs.