Repression by the cI protein of phage λ: in vitro inhibition of RNA synthesis

We have studied the in vitro repression of RNA synthesis by the cI protein of phage λ. We find that highly purified cI protein is an effective and specific repressor of RNA synthesis from the early gene region of λ DNA. Under optimal conditions at least 95% of the early gene RNA synthesis is repressed and this repression is eliminated or severely impaired by the use of λ DNA-carrying operator-type mutations which reduce the binding affinity of the cI protein. Highly effective repression can be demonstrated only through the use of the initiation-inhibitor rifampicin, which presumably, selects “properly” initiated RNA chains; thus we can by-pass in vitro but not yet solve the problem of how the host polymerase initiates specifically in vivo from the immediate-early promoter sites.     

RNA Synthesis Startpoints in Bacteriophage λ: Are the Promoter and Operator Transcribed?

Hybridization and fingerprint analysis of in vitro synthesized λ RNA shows that four chains are initiated at sites corresponding to those seen in vivo and that each molecule starts with a specific sequence. In one case examined, the major leftward operon, the promoter and operator and not transcribed into RNA.     

Structure of poliovirus replicative intermediate RNA: Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4′-aminomethyl-4,5′,8-trimethylpsoralen. After purification from infected cells, undenatured RI² appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [³H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.     

Structure and Function of Informofers

DNA-like RNA (dRNA) is found in the nucleus as a complex with globular protein particles called informofers. Intact informofers may be detached from the dRNA and can form complexes in vitro and in vivo with different kinds of dRNA, including virus-specific RNA synthesized in adenovirus-infected cells.     

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

Initial Nucleotide Frequencies of Bacterial RNA synthesized during Amino-acid Starvation or Changes of Carbon Source

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo¹ or in the proper conditions in vitro^2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine⁴. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.