Meredith Barb Homework-2 (25 points) Due on Sept.

24 by 5:00pm

1. Find the correct match. Items at the right could be used more than once. 2.) Subunit of the RNA polymerase holoenzyme that is responsible for the specificity of the polymerase 3.) Factor dependent termination 2.) Subunit of the RNA polymerase holoenzyme that is lost as it converts to an elongation complex 1) Capable of multiple catalytic and non-catalytic function because of its ability to form secondary structures 6.) DNA region where the RNAP binds to initiate transcription 7.) Factor independent termination 1. RNA

2. Sigma Factor 3. Rho

4. Ribosomal binding site

5. DNA 6. Promoter 7. GC-rich inverted repeat and Poly T tract

2. TrpR negatively regulates transcription of the tryptophan biosynthesis operon in the presence of tryptophan. a) What is the effector molecule for TrpR? The effector molecule binds to the repressor and allows it to bind to the operators is called the corepressor. For TrpR, this effector molecule is tryptophan. b) When is TrpR referred to as an aporepressor? A repressor that negatively regulates a biosynthetic operon is not active in the absence of the corepressor and in this state it is called an aporepressor. The TrpR aporepressor protein is the protein in the absence

of tryptophan, and when tryptophan binds to the aporepressor TrpR protein, it changes its conformation allowing it to bind to the operator. c) Describe another form of regulation that acts upon the tryptophan operon in the absence of TrpR (it involves the leader polypeptide). The trp operon is also subject to a completely different type of regulation called attenuation. Attenuation works by terminating transcription, which begins normally, before RNA polymerase reaches the first structural gene of the operon. After RNA Polymerase initiates transcription at the promoter, it moves through the trpL (leader region) to a site located just after region 2, where it then pauses. The hairpin formed by regions 1 and 2 is an important part of the signal to pause. This pause is short but it ensures that the ribosome has time to load onto the mRNA before RNA polymerase proceeds. The process of ribosome translation through the trp codons of trpL then determines whether a 3:4 hairpin will form which will cause termination, or another possible hairpin forming between 2:3, which would prevent the formation of the 3:4 hairpin. A 3:2 hairpin will form in the presence of low tryptophan levels. If the ribosome does not stall at the trp codons, it will continue until it reaches the UGA stop codon at the end of trpL, and while it remains at the stop codon while region 4 is synthesized, the ribosome will prevent a 2:3 hairpin from forming, therefore a 3:4 hairpin can form terminating transcription.

3. You are starting a new project in genomics and you are looking for small RNAs. You have a meeting with an expert in computers and programming. Unfortunately this person does not have experience in genomics. However he can do any kind of program you want. The program must have specific criteria to hunt small RNAs. What kind of features does the program have to look for in the sequences in order to identify small RNAs? A non-coding RNA is any region of RNA that doesnt encode a protein, and in bacteria these RNAs are often called small RNAs (sRNA). It is difficult to identify small RNAs by sequence inspection alone. Instead it is much easier to use the high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome. The existence distinct new RNA species can be confirmed by Northern analysis. Of the RNA determined by Northern analysis a minimal amount will encode ORFs, whereas the majority are likely to be novel functional small RNAs. Many of these small RNAs interact with the RNA-binding protein Hfq, pointing to a global role of the Hfq protein in facilitating small RNA function. That said , it is obviously difficult to identify sRNA by a program sequence

alone as the results would most likely be inaccurate, but if this was the only type of program available certain sequences would be the best place to look. Termination transcription sites would be a useful place to look for sRNAs. Factor-independent termination is characterized by an inverted repeat which is followed by a short run of AAAAs, meaning that the RNA transcribed from this DNA has a string of UUUUUUs, and this poly U sequence would be a great place to look for sRNA because transcription would be terminated at that point, and wouldnt begin again until another promoter region. There is also factordependent termination, more specifically p-dependent termination which has specific termination sites. These sites are termed rut ( for rho utilization sites) and are not easily identified but they do have a lot of CCCCs and little secondary structure. 4. What regulatory conditions must exist to allow expression of the lactose operon and why? (Will Glucose or Lactose be present and what effect will this have on the regulatory proteins in the cell) The lac operon is a classic example of negative regulation. The lac operon encodes the enzymes necessary for the utilization of lactose. The lac operon includes lacZ and lacY which are the structural genes. Another gene is now known which is lacA. These genes are only expressed in the presence of lactose. In the absence of lactose, the repressor binds to the operator sequence close to the promoter and thereby prevents RNA polymerase from binding, thus preventing transcription of the structural genes. In contrast, when lactose is present, the inducer binds the repressor and changes its conformation so that it can no longer bind to the operator sequence. However, if lactose is present but a better carbon/sugar source is present, such as glucose then the cell will utilize this better sugar instead and the structural genes will not be transcribed, this is called catabolite repression, and involves a transcriptional activator protein (CAP) and a small molecule effector molecule, along with cyclic AMP (camp).

5. What is helix-turn-helix motif? What is its biological function? Give one example (a protein) that has this motif. Most transcriptional regulation occurs at the level of transcription initiation at the promoter. This type of regulation occurs through proteins called transcriptional regulators, which usually bind to the DNA through helix-turnhelix motifs. In this motif a region of 7 to 9 amino acids forms an alpha-helical structure called helix 1. Helix 1 is separated by about 4 amino acids from another alpha-helical region of 7 to 9 amino acids called helix 2. These to helices are approximately right angles to each other, which explains the

terminology helix-turn-helix. When the protein binds DNA, helix 2 lies in the major groove of the DNA double helix, while helix 1 lies crosswise to the DNA. Since they lie in the major groove of DNA, the amino acids of helix 2 can contact and form specific Hydrogen-bonds with specific bases in the DNA. DNA-binding proteins containing this motif recognize and bind to specific regions of the DNA; many of the proteins exist as dimers and bind to inverted repeated DNA sequences. In these cases the two polypeptides are arranged head to tail so that the amino acids in helix 2 of each polypeptide can make contact with the same bases in the inverted repeats. An example of this motif is illustrated in the TrpR repressor protein. TrpR repressor is a dimmer and each copy of the trpR polypeptide has alphahelical structures. The helices of the aporepressor dimer are inactivate without trytophan bound not allowing the helix-turn-helix motif, but when the effector molecule tryptophan binds the repressor is activate and is able to bind to the operator with its active HTH domains which fit in the major groove forming the motif.

6. Describe how non-coding RNA (ncRNA) can increase and decrease the expression of a protein at the level mRNA stability. The majority of the human transcriptome is RNA that does not code for a protein called non-coding RNA (ncRNA). The function of ncRNA includes regulating the rate and timing of protein synthesis. Gene expression is tightly controlled at various levels, and critical cis-regulatory elements for posttranscriptional control are encoded in the 5' and 3' untranslated regions (UTRs). Many conserved sequence elements in UTRs have been identified as binding sites for proteins or antisense RNAs, which contribute to the regulation of nucleocytoplasmic transport, subcellular localization, translation and the stability of mRNAs. In translation initiation, the ribosome binds to the 5'-terminal cap of an mRNA and starts scanning the mRNA until it detects the first AUG start codon, where it initiates translation. The 5' UTRs contain binding sites for components of multiprotein transcription complexes and also participate in the recruitment of the 30S ribosomal subunit and translation initiation. The length of 5' UTRs, and the presence of additional upstream transcription start codons, is important for regulating the basal translation level of an mRNA, It has been shown that transcripts with an optimal start codon context tend to have shorter 5' UTRs, whereas an increased length of 5' UTR correlates with a 'weak' start codon context and with the presence of additional upstream start codons. A reduced level of translation also corresponds with the presence of minor open reading frames located within 5' UTRs and upstream of the main start codon in some genes.

7. Briefly describe the function of the tmRNA. The tmRNAs are a recently discovered 4th class of RNA, which can also be called SsrA or 10 Sa RNA. TmRNA is highly conserved and serves as a quality control mechanism. The dual nature of tmRNA (translation/messenger) is important in ribosome stalling, and makes it able to ass a peptide tag to the C-terminus of the unfinished protein, and targets said unfinished protein for proteolysis. MRNAs which lack a stop codon cause major problems, one of which is the fact that the protein will never be finished and thus will accumulate too much protein, which is toxic to the cell, and tmRNA helps this problem. This stable RNA is involved in releasing ribosomes that are unable to complete protein synthesis on mRNA lacking the stop codon. The resulting abnormal proteins are rapidly degraded by specific proteases, which recognize a signal peptide encoded by the template region of tmRNA. The discovery of trans-translation has caused a particular interest in structural and functional studies of tmRNA. 8. Explain how a polysome and Rho collaborate in creating a Rho-dependant polar effect in response to a stop codon introduced into an early gene of an operon. A polysome is multiple ribosomes translating a single mRNA. Polycistronic mRNA can create different problems, one of which is its polar effect on gene expression. Since translation and transcription are coupled, a nonsense codon in mRNA will stop translation, but RNA polymerase will not stop transcription forming a single-stranded uncoupled strand. When translation ceases at the nonsense codon upstream, the rut (rho utilization site) sequence, which is normally hidden, will become exposed. Rho-dependent termination then begins because this type of factor dependent termination only occurs if RNA is not being translated. Rho factor is then able to chase down the RNA polymerase, and once rho has caught up , its helicase activity is able to unstable and release the RNA-DNA hybrid, thus terminating transcription.