PRIMER When we dissect chromatin, we arrive at the highest resolution, which is the DNA wrapped around histones. The structure of DNA, the double helix, needs no elaborate introduction. In this chapter, the basic elements that contribute to the 3-D structure of DNA are presented. The helical DNA can, in fact, assume different structural forms, and we discuss them along with their significance. However, not all DNA exists as double-stranded. At the ends of the chromosomes, DNA is single-stranded and is characterized by a specific nucleotide sequence. The ends of the chromosomes are called telomeres, and they can assume a distinct 3-D structure. This structure is presented along with the structure of proteins that binds telomeric sequence and plays roles in their stability that eventually affect the length of chromosomal DNA and its role in replication and cell division.

PRIMER Transcription in eukaryotes is more complicated than in prokaryotes. In eukaryotes, there must be tissue-specific gene transcription because many different cell types need to be created. Therefore, transcription must be selective in different cells and tissues. This selectivity is possible by the cell- or tissue-specific transcriptional factors. These factors can bind sequences present in promoters or enhancers of genes that will be specifically expressed in a particular cell type. The basal transcriptional machinery, which is assembled at the promoters of genes and is responsible for the recruitment of RNA polymerase, is also more complex in eukaryotes. In addition, in eukaryotes there are three RNA polymerases, each one transcribing a particular set of genes.

As in Chapter 5, I begin this chapter by presenting the 3-D structure of eukaryotic RNA polymerase II, which transcribes the genes that produce mRNAs. A comparison is made with the prokaryotic RNA polymerase. I then describe the factors that constitute the basal transcriptional machinery, their 3-D structure, and their interaction with DNA, RNA polymerase II, and each other. The reader should be able to obtain a very informative picture of this assembly that leads to the initiation of eukaryotic transcription. I then proceed to the regulation of transcription. As I mentioned earlier, regulation of transcription is unique for each of the more than 200 different cell types. Covering everything we know about regulation is, therefore, overwhelming.

PRIMER DNA replication involves many proteins acting at different steps. DNA polymerases are the main enzymes needed to create new strands from the parental ones. However, DNA polymerase alone cannot complete the whole task of replication. When the double-stranded DNA melts at the origin of replication to expose the single strands to the polymerizing enzyme, the fork must continue to unwind until the termination signals have been reached. This part in replication is performed by helicases, and the topology ahead of the fork (mainly coiling and superwinding of the DNA) is controlled by topoisomerases. Finally, termination is achieved by the interaction of both helicases and termination proteins. Editing is another important function during replication to ensure that misincorporations are eliminated and, thus, mutations are avoided. Also, other proteins are involved in securing the processivity of DNA polymerase and the processing of the Okazaki fragments. The 3-D structure of all the major players participating in the entire process of replication is for the most part known. For some of them, we also know how they interact with each other or with DNA. This enables us to visualize replication in a 3-D journey. This chapter introduces the 3-D structure of DNA polymerases, the mechanism of incorporation of the incoming nucleotide, and how DNA polymerase interacts with DNA and other proteins to achieve processivity and editing. We then examine the structure of different helicases, their interaction with DNA, and the mechanism of unwinding as suggested by the 3-D structures.

PRIMER Transcription produces an RNA transcript, which contains the entire nucleotide sequence as it is imprinted in the DNA. In other words, the primary RNA transcript also contains the intronic sequences. These intronic sequences must be removed to create a continuous coding sequence that will be able to be translated to a protein. The process of intron removal is called splicing. However, not all introns are spliced-out the same way. Depending on the mechanism involved in their splicing, the introns are divided into three groups. In this chapter, I will present the steps of intron removal, emphasizing the 3-D structure of RNAs and proteins involved. Some introns are removed with the help of proteins, but others self-catalyze their cleavage. Therefore, the 3-D structure of the introns must be important in splicing and will be presented in order to visualize the mechanism. The determination of the 3-D structure of self-catalyzed introns is another celebrated example of how the structure reveals the function.

PRIMER As the polypeptide is born and exits from the ribosome, it is bound by factors that will enable it to pass through the membranes of the endoplasmic reticulum. This is possible because all secreted proteins contain a signal in the very N-terminus, the signal peptide. This is recognized by the signal recognition particle that brings it to the receptor in the membrane and facilitates the passing. Then, the polypetide has to fold into its unique 3-D structure. We already know how important this is. The function of a protein depends on its 3-D structure. The folding must be correct; otherwise, the protein will not be allowed to perform its tasks. Folding involves many factors, called chaperones. The 3-D structures of chaperones also provide stunning images of the mechanism of folding. Alas, nothing lives forever! And this rule applies to proteins as well. In fact, it is central to regulation that the protein will die after some time. If a protein does not die, it will accumulate and induce diseases. Eventually, aged proteins will be recognized and brought to their demise with the help of particular proteins and structures. These processes are examined and compared in prokaryotes and eukaryotes. It will be the end of the protein, the end of our journey, and the end of the book.

PRIMER When the mRNA is in the cytoplasm, it is recognized by factors and complexed with the ribosome, which is the place where the message is decoded and the protein is built up. We are rather fortunate because most of the players participating in protein synthesis are known at the 3-D level; thus, we can reconstruct all the steps of protein synthesis at the 3-D level. Some of the figures, especially the ones depicting the process of protein synthesis with the ribosome, are so stunning that I virtually feel I am there viewing the process first hand. For this spectacle to flow in the most comprehensive way possible, I selected a certain sequence for the presentation. First, I present the 3-D structure of the two major players in protein synthesis: tRNA (alone and complexed with aatRNA synthetase) and ribosome. The reader must become very familiar with both, especially with the ribosome. Even though we know the structure of the ribosome in high resolution, I first introduce its structure by showing a series of images from low to high resolution. This approach should enable the reader to understand the structure better. Having introduced the basic players, I proceed with the first stage of protein synthesis, the initiation. This is compared in both prokaryotes and eukaryotes, and the known 3-D structure of initiation factors and their interaction with rRNA are presented.

PRIMER Unwinding of DNA is not only restricted during replication but also occurs during transcription, even though the unwinding takes place in a different way. Transcription is a very important step, perhaps the most important step in regulation. Transcription determines which genes will be expressed and, therefore, proceed for decoding and protein synthesis. During the process of transcription, the 3′ → 5′ noncoding DNA strand becomes the template for the synthesis of RNA. The RNA transcript, therefore, keeps the sequence order of the coding strand, 5′ → 3′. The main enzyme here is RNA polymerase. Regulation at this level is very unique and markedly different in prokaryotes and eukaryotes. Therefore, for clarity, I describe transcription in prokaryotes first (this chapter) and continue with transcription in eukaryotes in Chapter 6).

The first part of this chapter examines the structure of the prokaryotic RNA polymerase and the mechanisms of transcription initiation and elongation as revealed by 3-D complexes of RNA polymerase and DNA. In the second part, I present some of the mechanisms involved in regulating gene transcription in prokaryotes. Having as a focus to present these events at the 3-D level, my aim is not to be redundant, and in doing so I have omitted information that can be found in microbiology and biochemistry books. Therefore, the basic structural motifs involved in transcriptional regulation are presented in a few regulators to pinpoint similarities and differences.

PRIMER The three-dimensional structure of nucleic acids and proteins as it pertains to the mechanisms involved in gene regulation is the major focus of this book. Therefore, the reader will encounter many 3-D structures. The first chapter of the book presents the very basic ideas behind the three-dimensional aspects of biomolecules. The first part deals with the techniques used to determine 3-D structures. The presentation is virtually for the layperson. Then the basic structural elements found in proteins are examined. Having done this, we examine a particular 3-D structure (that includes both DNA and protein) presented with different modeling. This exercise will help you to become familiar with the different ways that scientists present their 3-D structures. We use different models because one aspect of structure and function can be better represented with one model, whereas another aspect is more suited to a different model.

PRIMER DNA is not free in the nucleus; it is bound by the proteins that package it. This fact is important because DNA must be accommodated in a small place. Also, as we will see in Chapter 6, the packaging provides a level of transcriptional control. In this chapter, the goal is to become familiar with the proteins that are involved in packaging and their effects on DNA. The first part of the chapter examines the 3-D structure of the nucleosome and its components, the histones. This structure is the highest order of DNA packaging. Other proteins (nonhistone), however, have the ability to bend DNA and, therefore, provide another degree of organization. Such architectural proteins are important because linear DNA is not very efficient when it comes to regulation. Regulatory elements are often separated by long distances, and they must come close for interaction. Also, bending DNA makes it more accessible for interactions with proteins. These nonhistone proteins possess different structural motifs that mediate interaction with DNA. Their structural characteristics and effects on DNA are presented. The importance of these architectural proteins is also stressed in later chapters.

PRIMER The spliced and modified mRNA must be transported to the cytoplasm for translation that will result in protein synthesis, which is the ultimate goal of transcription. The question addressed in this chapter involves the degree to which the topology of transcription and movement of the transcripts toward the nuclear pores is organized. This area of inquiry is a relatively new, and experiments are possible only by using modern image analysis techniques. Evidence is presented that transcription and movement of transcripts are organized topologically in the nucleus. Such an organization is compared with DNA replication. This information is very useful in understanding the spatial employment of these events.