Transcriptional activation of bacteriophage T4 middle promoters by the motA protein.

Transcriptional activation of middle genes in bacteriophage T4 requires the phage-encoded motA protein. Many middle genes are involved in deoxyribonucleotide biosynthesis and phage DNA replication. In the absence of motA, the gene products that are required for DNA synthesis are transcribed from other, upstream promoters. Using primer extension sequencing on RNA templates isolated from T4 motA+ and motA- infected cells, we have characterized 14 motA-dependent transcripts. The T4 middle promoters have a consensus sequence of nine base-pairs, (a/t)(a/t)TGCTT(t/c)A, spaced 11 to 13 nucleotides away from the Escherichia coli--10 consensus sequence, TAnnnT. The motA protein also can act as a transcriptional repressor for at least one early gene. Furthermore, the phage-encoded motA protein can activate in trans a middle promoter resident on a plasmid.

[A new early gene in front of the middle gene 31 of bacteriophage T4: cloning and expression]. / Novyi rannii gen pered srednim genom 31 bakteriofaga T4: klonirovanie i ékspressiia.

We have identified a new gene, which we designated gene 31.1, as the nearest upstream neighbour of gene 31. We cloned the 1.03 k.b. EcoRI/BglII fragment of T4 DNA, and expressed both genes using the T7 RNA polymerase/promoter two plasmid system. Gene 31.1 encodes a protein with molecular weight of about 10 kDa, and the C-terminal portion of this gene is the incomplete open reading frame ORF 31.1 determined earlier. Using primer extension sequencing on RNA templates isolated from T4 mot A+ and mot A- infected cells, we have shown that gene 31 has a middle mode mot A-dependent transcript starting at two points and which is initiated from gene's 31 own promoter. Gene 31 is also transcribed from an early upstream promoter into a polycistronic mRNA which covers an early gene 31.1 as well.

Why peer discussion improves student performance on in-class concept questions.

When students answer an in-class conceptual question individually using clickers, discuss it with their neighbors, and then revote on the same question, the percentage of correct answers typically increases. This outcome could result from gains in understanding during discussion, or simply from peer influence of knowledgeable students on their neighbors. To distinguish between these alternatives in an undergraduate genetics course, we followed the above exercise with a second, similar (isomorphic) question on the same concept that students answered individually. Our results indicate that peer discussion enhances understanding, even when none of the students in a discussion group originally knows the correct answer.

DNA polymerase of bacteriophage T4 is an autogenous translational repressor.

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32) is, like gp43, autogenously regulated at the translational level.

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins. Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.