Genetics 2450 CH.5

Discuss the similarities and differences between E.coli RNA polymerase and Eukaryotic RNA polymerases.

Both Eukaryotic and E.coli RNA polymerases transcribe RNA in a 5' to 3' direction using a 3' to 5' DNA template strand. There are many differences between the enzymes, however. In E.coli, a single RNA polymerase core enzyme is used to transcribe genes. In eukaryotes, there are three types of RNA polymerase molecules: RNA polymerase I, II, and III. RNA polymerase I synthesizes 28S, 18S, and 5.8S rRNA and is found in the nucleolus. RNA polymerase II synthesizes hnRNA, mRNA, and some snRNAs and is nuclear. RNA polymerase III synthesizes tRNA, 5S rRNA and some snRNAs and is also nuclear. Each RNA polymerase uses a unique mechanism to identify those promoters at which it initiates transcription. In prokaryotes a sigma factor procides specificity to the sites bound by the four-polypeptide core enzyme, so that it binds to promoter sequences. The holoenzyme loosely binds to a sequence lying about 35 bp before transcription initiation changes conficuration, and then tightly binds to a region about 10 bp beofre transcription initiation and melts about 17 bp of DNA around this region. The two-step binding of the promoter orients the polymerase on the DNA and facilitates transcription initiation in the 5' to 3' direction. After about 8-9 bases are formed in the new transcript, sigma factor dissociates from the holoenzyme and the core enzyme completes the transcription process. Although the principles by which the eukaryotic RNA polymerases bind their promoters are similar in that they use a set of ancillary protein factors-transcription factors-the details are quite different. IN eukaryotes, each of the three types of RNA polymerases recognizes a different set of promoters by using a polymerase specific set of transcription factors, and the mechanisms of interaction are different.

Compare DNA and RNA with regard to their structure, function, location, and activity. How do these molecules differ with regard to the polymerases used to synthesize them?

Both are composed of linear polymers of nucleotides but have different bases-DNA has Thymine and RNA has Uracil. DNA is also made of deoxyribose sugar while RNA is made of ribose. DNA is generally double stranded while RNA is single stranded. DNA is wound in a double helix and packaged by proteins into chromosomes, either as nucleoid body in prokaryote or nucleus in eukaryote. RNA can form stable, function stem-loop structures as in tRNA. RNA can be exported into cytoplasm and if it is mRNA it can be bound by ribosomes and translated. DNA functions as a storage molecule, while RNA functions as a messenger, in translation, and in eukaryotic RNA processing. Both DNA and RNA polymerases catalyze the synthesize of nucleic acid in a 5' to 3' direction. Both use DNA template and synthesize complementary nucleic acid polynucleotide. DNA requires 3'OH to add onto and RNA does not. RNA can initiate chains without primers while DNA polymerase can't. RNA polymerase require specific base pair sequences to initiate transcription.

What are the most significant differences between the organization and expression of bacterial genes and eukaryotic genes?

Prokaryotic and eukaryotic genes differ in their gene structure, in their RNA processing and in how transcription is couples to translation. Prokaryotic gens are defined by an upstream promoter, and RNA coding sequence, and a downstream terminator. Eukaryotic genes have these features, but the promoters are much more complex, and nearby or distant enhancer and silencer elements can strongly affect the level of transcription of eukaryotic genes. The RNA coding sequences of eukaryotes can be interrupted with introns. Finally, prokaryotic mRNAs are often polcistronic containing the amino acid coding information for more than one gene. Eukaryotic mRNAs are generally monocistronic containing the amino acid coding information for only one gene. A notable exception is in C. elegans where polycistronic mRNAs are found. Prokaryotic genes lack introns, while eukaryotic genes typically have one or more introns; therfore, a transcribed region can be larger than the size of a mature mRNA. THe excision of introns from primary mRNAs is only one aspect of the processing of eukaryotic RNAs. In addition eukaryotic mRNAs ar emodifed in both their 5' and 3' ends: They are capped and plyadenylated. Since prokaryotes lack a nucleus, transcription is directly coupled to translation. In eukaryotes, mRNAs must be processed and then transcported out of the nucleus before they are translated by ribosomes in the cytoplasm. These three differences provide cell types in a multicellular eukaryote with three means to regulate gene expression. Gene expression can be regulated at each of these three levels: transcription, mRNA processing, and translation.

Discuss the molecular events involved in the termination of RNA transcriptase in bacteria. IN what ways is the process fundamentally different in Eukaryotes.

Termination of transcription in E.coli is signaled by controlling elements (sequences within the DNA) called terminators. Two classes of terminators exist, rho-independent (Type I) and rho-dependent (Type II). Both Type I and Type II termination ends in the cessation of RNA synthesis and the release of both the RNA chain and RNA polymerase from the DNA. Type I terminators utilize the sequences with twofold symmetry lying about 16-20 bp upstream of the termination point to signal the termination site. The hairpin loop that forms with a twofold symmetric sequence is transcribed, plus a string of Us, lead to termination, perhaps by destabilizing the RNA-DNA hybrid in the terminator region. Type II terminators lack the structure of Type I terminators and instead use an ATP activated rho protein that binds to recognition sequences in the transcribed termination region. The binding of rho leads to the hydrolysis of the ATP and the release of the transcript and the RNA polymerase from the DNA template. IN eukaryotes, transcription termination differs depending on the RNA polymerases under consideration. RNA polymerase I transcribes repeated 18S, 5.8 S, and 28S rDNA clusters as single transcription units. Transcription of each unit is terminated at a specific site. THe termination site lies in the nontranscribed spaces (NTS) between adjacent rDNA transcription units. RNA polymerae II transcribes genes for mRNAs and some snRNAs. Transcription termination for these genes is fundamentally different from transcription termnation in bacteria because these genes lack specific transcription termination sequences at their 3' ends. mRNA transcription can continue for hundreds or thousands of nucleotides downstream of the protein-coding sequence until it is past a poly(A) site in the RNA. The poly(A) site is recognized and cleaved by a complex set of proteins. It is positioned 10 to 30 nucleotides after an AAUAAA sequence and is followed by a GU rich or U rich sequence. Once the RNA is cleaved, poly(A) polymerase adds A nucleotides onto the 3'OH of the RNA to produce a poly(A) tail. RNA polymerase III transcribes genes for 5S rRNA, tRNA, and some snRNAs. Termination events for RNA polymerase III were not discussed in this chapter.

All base pairs in the genome are replicated during the DNA synthesis phase of the cell cycle, but only some of the base pairs are transcribed into RNA. How is it determined which base pairs of the genome are transcribed into RNA?

The DNA sequences that are transcribed into RNA are determined using two general principles. First, signals in the DNA base sequences identify the specific region to be transcribed. Only regions bounded by transcription initiation and transcription termination signals are transcribed. In regions bounded by these signals, only one strand is ordinarily transcribed, so that transcripts are formed in a 5' to 3' direction using a single DNA template strand. Second, transcription within a defined region occurs only if additional transcription-inducing molecules are present. Some of these molecules recognize the signals within the DNA that define the transcription unit and are used in the transcription process.