Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 245

1 RNA is a polymer made up of a chain of ribonucleotides. The bases of the nucleotides—adenine, cytosine, uracil, and guanine—are atiached to the five-carbon sugar ribose. Phosphate bonds connect the sugars to make the RNA chain, attaching the third (3′) carbon of one sugar to the fifth (5′) carbon of the next sugar. The 5′ end of the RNA is the nucleotide that has a free phosphate attached to the 5′ carbon of its sugar. The 3′ end has a free hydroxyl group at the 3′ carbon, with no phosphate attached. RNA is both made and translated from the 5′ end to the 3′ end.

2 After they are synthesized, RNAs can undergo extensive processing and modification. Processing occurs when phosphate bonds are broken or new phosphate bonds are formed. Modification occurs when the bases or the sugars of the RNA are chemically altered, for example, by methylation. In bacteria, rRNAs and tRNAs, but not mRNAs, are extensively modified.

3 The primary structure of an RNA is its sequence of nucleotides. The secondary structure is formed by hydrogen bonding between bases in the same RNA to give localized doublestranded regions. The tertiary structure is the three-dimensional shape of the RNA due to the stiffness of the double-stranded regions of the secondary structure. All RNAs, including mRNA, rRNA, and tRNA, probably have secondary and tertiary structures.

4 The enzyme responsible for making RNA is called RNA polymerase. One of the largest enzymes in the cell, the bacterial RNA polymerase core enzyme has five subunits plus another detachable subunit, the σ factor, which comes off after the initiation of transcription. The ω subunit helps in its assembly. The core enzyme is active in transcription elongation but requires addition of σ to form the holoenzyme to initiate transcription.

5 Transcription begins at well-defined sites on DNA called promoters. The type of promoter used depends on the type of σ factor bound to the RNA polymerase.

6 Transcription stops at sequences in the DNA called transcription terminators, which can be either factor dependent or factor independent. The factor-independent terminators have a string of A’s (transcribed as U’s in the RNA) that follows a sequence that forms an inverted repeat. The RNA transcribed from that region folds back on itself to form a stem-loop, or hairpin, which causes the RNA molecule to fall off the DNA template. The factor-dependent terminators do not have a well-defined sequence. The ρ protein is the best-characterized termination factor in E. coli. It forms a ring that binds to and encircles the RNA, moving toward the RNA polymerase. If the RNA polymerase pauses at a ρ termination site, the ρ factor catches up to it and causes it to dissociate from the DNA and release the RNA product.

7 Most of the RNA in the cell falls into three classes: messenger (mRNA), ribosomal (rRNA), and transfer (tRNA). mRNA is very unstable, existing for only a few minutes before being degraded. rRNAs in bacteria are further divided into three types: 16S, 23S, and 5S. Both rRNA and tRNA are very stable and account for about 95% of the total RNA. Other RNAs include the primers for DNA replication and small RNAs involved in regulation or RNA processing.

8 Ribosomes, the site of protein synthesis, are made up of two subunits, the 30S subunit and the 50S subunit, which contain the rRNAs as well as approximately 50 proteins. The 16S rRNA is in the 30S subunit, while the 23S and 5S rRNAs are in the 50S subunit.

9 Polypeptides are chains of the 20 amino acids that are held together by peptide bonds between the amino group of one amino acid and the carboxyl group of another. The amino terminus (N terminus) of the polypeptide has the amino acid with an unattached amino group. The carboxy terminus (C terminus) of a polypeptide has the amino acid with a free carboxyl group.

10 Translation is the synthesis of polypeptides, using the information in mRNA to direct the sequence of amino acids. During translation, the mRNA moves in the 5′-to-3′ direction along the ribosome 3 nucleotides at a time. Three reading frames are possible, depending on how the ribosome is positioned at each triplet.

11 The genetic code is the assignment of each possible 3-nucleotide codon sequence in mRNA to 1 of 20 amino acids. The code is redundant, with more than one codon sometimes encoding the same amino acid. Because of wobble, the first position of the tRNA anticodon (written 5′ to 3′) does not have to behave by the standard base-pairing complementarity to the third position of the antiparallel codon sequence, and other pairings are possible.

12 Initiation of translation occurs at TIRs on the mRNA that consist of an initiation codon, usually AUG or GUG, and often an S-D sequence, a short sequence that is complementary to part of the 16S rRNA and precedes the initiation codon.

13 In bacteria, the first tRNA to enter the ribosome is a special methionyl-tRNA called fMet-tRNAfMet, which carries the amino acid formylmethionine. After the polypeptide has been synthesized, the formyl group and often the first methionine are removed.

14 Translation termination occurs when one of the termination or nonsense codons, UAA, UAG, or UGA, is encountered as the ribosome moves down the mRNA. Proteins called release factors are also required for release of the polypeptide.

15 The primary structure of a polypeptide is the sequence of amino acids in the polypeptide. Proteins can be made up of more than one polypeptide chain, which can be the same as or different from each other. The secondary structure results from hydrogen bonding of the amino acids to form α-helical regions and β-sheets. Tertiary structure refers to how the chains fold up on themselves, and quaternary structure refers to one or more different polypeptide chains folding up on each other.

16 Proteins that help other proteins fold are called chaperones. The most ubiquitous chaperones are the Hsp70 chaperones, called DnaK in E. coli, which are very similar in all types of cells from bacteria to humans. These chaperones bind to the hydrophobic regions of proteins and prevent them from associating prematurely. They are aided by their smaller cochaperones, DnaJ and GrpE, which help in binding to proteins and cycling ADP off the chaperone, respectively. Other proteins, called Hsp60 chaperonins, also help proteins fold, but by a very different mechanism. One chaperonin, called GroEL in E. coli, forms large cylindrical structures with internal chambers that take up unfolded proteins and help them refold properly. A cochaperonin called GroES forms a cap on the cylinder after the unfolded protein is taken up. Chaperonins like GroEL are found in bacteria and in the organelles of eukaryotes and are called group I chaperonins. Another type, group II chaperonins, is found in the cytoplasm of eukaryotes and in archaea. They have a similar structure but a very different amino acid sequence.

17 The process of passing proteins through membranes is called transport. Proteins that pass through the inner membrane into the periplasm and beyond are said to be exported. Proteins that pass out of the cell are secreted.

18 Proteins can also be held together by disulfide linkages between cysteines in the protein. Generally, only proteins that are exported into the periplasm or out of the cell have disulfide bonds. These disulfide bonds are made by oxidoreductases in the periplasm of bacteria with an outer membrane.

19 Bacterial cells utilize a variety of mechanisms to target proteins to different locations, such as the cell membrane, the periplasm, or outside the cell. Some of these systems are used for a wide range of proteins, while others are specific to individual sets of proteins.

20 The expression of genes is regulated, depending on the conditions in which the cell is found. This regulation can be either transcriptional or posttranscriptional. Transcriptional regulation can be either negative or positive, depending on whether the regulatory protein is a repressor or an activator, respectively. A repressor binds to an operator, which is usually close to the promoter, and prevents transcription from the promoter. An activator binds to an activator sequence that is usually upstream of the promoter and increases transcription from the promoter. Transcriptional regulation can also occur after the RNA polymerase leaves the promoter, as in attenuation or antitermination of transcription. Posttranscriptional regulation can occur at the level of stability of the mRNA, translation of the mRNA, or processing, modification, or degradation of the gene product.

21 The strand of DNA from which the mRNA is made is the transcribed, or template, strand. The opposite strand, which has the same sequence as the mRNA, is the coding, or nontemplate, strand.

22 A sequence 5′ on the coding strand of DNA relative to a particular element is said to be upstream, whereas a sequence 3′ to that element is downstream.

23 The TIR sequence of a gene does not necessarily occur at the beginning of the mRNA. The 5′ end of the mRNA is called the 5′ untranslated region or leader region. Similarly, the sequence downstream of the termination codon is the 3′ untranslated region.

24 Because mRNA is both transcribed and translated in the 5′-to-3′ direction, translation can begin before synthesis of the mRNA is complete in bacteria, which have no nuclear membrane.

25 Bacteria and archaea often make polycistronic mRNAs with more than one polypeptide coding sequence on an mRNA. This can result in polarity of transcription and translational coupling, phenomena unique to these domains, where mutations in the 5′ coding region of an mRNA can affect the expression of genes in the 3′ region.

26 An ORF is a string of amino acid codons in DNA unbroken by a termination codon. In vitro transcription-translation systems or transcriptional and translation fusions are often required to prove that an ORF in DNA actually encodes a protein.

27 Gene fusions have many uses in modern molecular genetics. They can be either transcriptional or translational fusions. In a transcriptional fusion, the downstream reporter gene is transcribed onto the same mRNA as the upstream gene, but the reporter gene coding region is translated from its own TIR, so expression of the downstream reporter gene is dependent on the activity of the promoter of the upstream gene but not the translational signals of the upstream gene. In a translational fusion, the two coding regions are fused to each other, so expression of the downstream reporter gene is dependent on the activities of both the promoter and TIR of the upstream gene.

28 Many naturally occurring antibiotics target components of the transcription and translation machinery. Some of the most commonly used are rifampin, streptomycin, tetracycline, thiostrepton, chloramphenicol, and kanamycin. In addition to their uses in treating bacterial infections, tumor chemotherapy, and biotechnology, antibiotics have also helped us understand the mechanisms of transcription and translation. In addition, the genes that confer resistance to these antibiotics have served as selectable genetic markers and reporter genes in molecular genetic studies of organisms in all domains of life.