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.
QUESTIONS FOR THOUGHT
1 Which do you think came first in the very earliest life on Earth, DNA, RNA, or protein? Why?
2 Why is the genetic code universal?
3 Why do you suppose prokaryotes have polycistronic mRNAs but eukaryotes do not?
4 Why do you suppose mitochondrial genes show differences in their genetic code from chromosomal genes in eukaryotes?
5 Why is selenocysteine inserted into proteins of almost all organisms but into only a few sites in a few proteins in these organisms?
6 Why do so many antibiotics inhibit the translation process as opposed to, say, amino acid biosynthesis?
7 Why do you think chaperonins have two linked chambers and alternate the folding of proteins between the two chambers?
8 List all the reasons you can think of why bacteria would regulate the expression of their genes.
9 Why do bacteria have so many different mechanisms for localization of proteins to different sites?
SUGGESTED READING
1 Agashe VR, Guha S, Chang H-C, Genevaux P, Hayer-Hartl M, Stemp M, Georgopoulos C, Hartl FU, Barral JM. 2004. Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117:199–209.
2 Bae B, Feklistov A, Lass-Napiorkowska A, Landick R, Darst SA. 2015. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife 4:e08504.
3 Baker TA, Sauer RT. 2006. ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci 31:647–653.
4 Ban N, Nissen P, Hansen J, Capel M, Moore PB, Steitz TA. 1999. Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit. Nature 400:841–847.
5 Becker SH, Darwin KH. 2017. Bacterial proteasomes: mechanistic and functional insights. Microbiol Mol Biol Rev 81:e00036–16.
6 Björk GR, Hagervall TG. 25 July 2005, posting date. Transfer RNA modification. EcoSal Plus 2005 doi:10.1128/ecosalplus.4.6.2.
7 Browning DF, Busby SJW. 2004. The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65.
8 Bukau B, Horwich AL. 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92:351–366.
9 Condon C. 2007. Maturation and degradation of RNA in bacteria. Curr Opin Microbiol 10:271–278.
10 Feilmeier BJ, Iseminger G, Schroeder D, Webber H, Phillips GJ. 2000. Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182:4068–4076.
11 Freudl R. 2013. Leaving home ain’t easy: protein export systems in Gram-positive bacteria. Res Microbiol